Methods of treating anaemia

ABSTRACT

The invention relates to human targets of interest (TOI), anti-TOI ligands, kits compositions and method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 of aco-pending U.S. application Ser. No. 14/978,183, filed Dec. 22, 2015,which is a continuation application under 35 U.S.C. §120 of a co-pendingU.S. application Ser. No. 14/868,952, filed Sep. 29, 2015, which is acontinuation application under 35 U.S.C. §120 of U.S. application Ser.No. 14/731,727 filed Jun. 5, 2015, issued as U.S. Pat. No. 9,187,562 onNov. 17, 2015, which is a continuation application under 35 U.S.C. §120of U.S. application Ser. No. 14/665,579 filed Mar. 23, 2015 issued asU.S. Pat. No. 9,139,648 on Sep. 2, 2015, which is a continuation-in-partapplication of U.S. application Ser. No. 14/331,730 filed Jul. 15, 2014,the entire contents of which are herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on created oncreated on Feb. 9, 2016, is named 069496-082417_SL.txt and is 623,759bytes in size.

TECHNICAL FIELD

The technology described herein relates to ligands, e.g., antibodies forthe treatment of disease.

BACKGROUND

It is recognized that individual humans differ in their sequence andrecently several individuals have had their genomes sequenced, forinstance James Watson and Craig Venter. Comparison of the genomesequence of individuals has revealed differences in their sequences inboth coding and non-coding parts of the genome. Some of these variationsin humans are significant and contribute to phenotypic differencesbetween individuals. In extreme cases these will result in geneticdisease. The 1000 Genomes Project has the objective of cataloguingsequences in the human genome, involving sequencing the genomes of avery large sampling of individuals from diverse art-recognized humanethnic populations.

SUMMARY

Through the application of human genetic variation analysis andrationally-designed sequence selection the present invention providesfor improved human patient diagnosis and therapy. Importantly, theinvention enables tailored medicines that address individual humanpatient genotypes or phenotypes.

The inventor's analysis of large numbers of naturally-occurring genomichuman TOI sequences reveals that there is significant variation acrossdiverse human populations and provides for the ability for correlationbetween individual human patients and tailored medical and diagnosticapproaches addressing the target. The technical applications of thesefindings, as per the present invention, thus contribute to bettertreatment, prophylaxis and diagnosis in humans and provides for patientbenefit by enabling personalized medicines and therapies. This providesadvantages of better prescribing, less wastage of medications andimproved chances of drug efficacy and better diagnosis in patients.

Furthermore, the inventor surprisingly realised that some rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention and better servingpatients in those populations.

With this, the inventor realised that there is significant industrialand medical application for the invention in terms of guiding the choiceof an anti-TOI ligand for administration to human patients for therapyand/or prophylaxis of TOI-mediated or associated diseases andconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

To this end, the invention provides:—

In a First Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   a. Administering to the human an anti-TOI ligand to target a TOI    variant in the human and treat or prevent said disease or condition,    wherein the TOI in said human is encoded by a nucleotide sequence    having a cumulative human allele frequency of less than 50% and/or    wherein the TOI in said human is encoded by a nucleotide sequence    having a total human genotype frequency of less than 50%;    -   wherein-   b. Before step (a) said human has been or is genotyped as positive    for said nucleotide sequence or phenotyped as positive for said TOI    variant.

In a Second Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is encoded by a        nucleotide sequence having a cumulative human allele frequency        of less than 50% and/or wherein the TOI in said human is encoded        by a nucleotide sequence having a total human genotype frequency        of less than 50%; wherein    -   b. before step (a) the ligand has been or is determined as        capable of binding to said TOI variant.

In a Third Configuration

A method of treating or preventing a disease or condition in a human,wherein the disease or condition is mediated by a Target of Interest(TOI), wherein the TOI is present in humans as different polymorphicvariants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is a variant encoded by        a nucleotide sequence having a cumulative human allele frequency        of more than 50% and/or having a total human genotype frequency        of more than 50%; wherein    -   b. Before step (a) said human has been or is genotyped as        negative for a variant nucleotide sequence having a cumulative        human allele frequency of less than 50% and/or having a total        human genotype frequency of less than 50%; or phenotyped as        negative for a TOI variant encoded by a nucleotide sequence        having a cumulative human allele frequency of less than 50%        and/or having a total human genotype frequency of less than 50%.

In a Fourth Configuration

An anti-human TOI ligand for use in a method of treating and/orpreventing a TOI-mediated disease or condition in a human, wherein theTOI is present in humans as different polymorphic variants and whereinthe genome of said human comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%, the method comprising administeringthe ligand to the human.

In a Fifth Configuration

A ligand that binds a human TOI comprising an amino acid sequenceencoded by a TOI nucleotide sequence having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50%, for use in a method comprising the step of using theligand to target said TOI in a human to treat and/or prevent a diseaseor condition mediated by TOI, the method comprising administering theligand to the human.

In a Sixth Configuration

A pharmaceutical composition or kit for treating or preventing acondition or disease mediated by a TOI.

In a Seventh Configuration

A method of producing an anti-human TOI antibody binding site, themethod comprising obtaining a plurality of anti-TOI antibody bindingsites, screening the antibody binding sites for binding to a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody binding site that binds inthe screening step.

In a Eighth Configuration

A method of producing an anti-human TOI antibody, the method comprisingimmunising a non-human vertebrate (eg, a mouse or a rat) with a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody that binds a TOIcomprising an amino acid sequence encoded by a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, and optionallyproducing a TOI-binding fragment or derivative of the isolated antibody.

In a Ninth Configuration

A kit for TOI genotyping a human, wherein the kit comprises a nucleicacid comprising a nucleotide sequence that specifically hybridises to aTOI nucleotide sequence selected having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50% or an RNA transcript thereof; and/or the nucleic acidcomprises a nucleotide sequence that comprises at least 10 contiguousnucleotides of a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50% or is an antisense sequence thereof.

In a Tenth Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortreating and/or preventing a TOI-mediated disease or condition in ahuman whose genome comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%.

In a Eleventh Configuration

Use of an anti-TOI ligand that binds a human TOI comprising an aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, in the manufacture of a medicament fortargeting said TOI in a human to treat and/or prevent a disease orcondition mediated by TOI.

In a Twelfth Configuration

A method of targeting a TOI for treating and/or preventing aTOI-mediated disease or condition in a human, the method comprisingadministering an anti-TOI ligand to a human comprising a TOI nucleotidesequence selected having a cumulative human allele frequency of lessthan 50% and/or a total human genotype frequency of less than 50%,whereby a TOI encoded by said nucleotide sequence is targeted.

In a Thirteenth Configuration

A method of TOI genotyping a nucleic acid sample of a human, the methodcomprising identifying in the sample the presence of a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

In a Fourteenth Configuration

A method of TOI typing a protein sample of a human, the methodcomprising identifying in the sample the presence of a TOI amino acidsequence encoded by a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or having a total human genotypefrequency of less than 50%.

In an example, the TOI is a human TOI selected from the group consistingof PCSK9, VEGF-A and IL6 receptor. In an example, the TOI is humanIL4Ra, PDGF-B, PDGFR-B or Ang-2.

A Fifteenth Configuration provides a ligand, method, use, kit orcomposition of the invention, wherein

-   -   (i) the ligand (eg, antibody or fragment) comprises    -   (a) a variable domain that is encoded by a human V region        nucleotide sequence, wherein the V nucleotide sequence is        derived from recombination of human VH, D and JH gene segments        or human VL and JL gene segments; or    -   (b) a constant region domain encoded by a C region gene segment;    -   Wherein a first gene segment of said gene segments of (a), or        said C region gene segment of (b) comprises a first single        nucleotide polymorphism (SNP) encoding a first amino acid        polymorphism; and    -   (ii) the genome of said human comprises said first SNP or        wherein said human expresses (a′) an antibody variable domain        comprising said first amino acid polymorphism or (b′) an        antibody constant domain comprising said first amino acid        polymorphism.

A Sixteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment that comprises a human antibody variabledomain derived from the recombination of a human V gene segment and ahuman J gene segment (and optionally a human D gene segment when thevariable domains are VH domains); and wherein the genome of the humancomprises said human V gene segment and/or the human expressesantibodies comprising antibody variable domains derived from therecombination of said human V gene segment and a human J gene segment(and optionally a human D gene segment).

A Sixteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused human PCSK9receptor) comprises a human heavy chain constant domain encoded by afirst constant region nucleotide sequence; and wherein the genome of thehuman comprises a heavy chain constant region nucleotide sequence thatis identical to said first constant region nucleotide sequence and/orthe human expresses antibodies comprising said human constant domain.

A Seventeenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human gamma-1 heavy chain constant region that comprises anAsp corresponding to position 204 of SEQ ID NO: 42 or a Leucorresponding to position 206 of SEQ ID NO: 42 and wherein the genome ofthe human comprises a gamma-1 heavy chain constant region nucleotidesequence that encodes such an Asp or Leu or the human expressesantibodies comprising human gamma-1 constant regions comprising such anAsp or Leu.

A Eighteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human gamma-2 heavy chain constant region that comprises anamino acid selected from the group consisting of a Pro corresponding toposition 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Valcorresponding to position 161 of SEQ ID NO: 44 and an Ala correspondingto position 257 of SEQ ID NO: 44; and wherein the genome of the humancomprises a gamma-2 heavy chain constant region nucleotide sequence thatencodes such a selected amino acid or the human expresses antibodiescomprising human gamma-2 constant regions comprising such a selectedamino acid.

A Ninteenth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand (eg, comprising orconsisting of an antibody or fragment or an Fc-fused TOI receptor)comprises a human kappa light chain constant region that comprises a Valcorresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding toposition 87 of SEQ ID NO: 50; and wherein the genome of the humancomprises a kappa light chain constant region nucleotide sequence thatencodes such a Val or Cys or the human expresses antibodies comprisinghuman kappa light chain constant regions comprising such a Val or Cys.

A Twentieth Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment, wherein the antibody or fragment comprises aVH domain that is derived from the recombination of a human VH genesegment, a human D gene segment and a human JH gene segment, wherein theVH gene segment is selected from the group consisting of (i) IGHV1-18*01and the genome of the human comprises a human IGHV1-18*01 nucleotidesequence or the human expresses antibodies comprising variable domainsderived from the recombination of human IGHV1-18*01; or (ii) IGVH1-46*01and the genome of the human comprises a human IGHV1-46*01 nucleotidesequence or the human expresses antibodies comprising variable domainsderived from the recombination of human IGHV146*01.

A Twenty-First Configuration provides the ligand, method, use, kit orcomposition of the invention, wherein the ligand comprises or consistsof an antibody or fragment, wherein the antibody or fragment comprises aVL domain that is derived from the recombination of a human VL genesegment and a human JL gene segment, wherein the VL gene segment isselected from the group consisting of (i) IGKV4-1*01 and the genome ofthe human comprises a human IGKV4-1*01 nucleotide sequence or the humanexpresses antibodies comprising variable domains derived from therecombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome ofthe human comprises a human IGLV2-14*01 nucleotide sequence or the humanexpresses antibodies comprising variable domains derived from therecombination of human IGLV2-14*01; or (iii) IGKV1-13*02 and the genomeof the human comprises a human IGKV1-13*02 nucleotide sequence or thehuman expresses antibodies comprising variable domains derived from therecombination of human IGKV1-13*02.

A Twenty-Second Configuration provides a method of treating or reducingthe risk of an IL4Ra-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human IL4RAprotein that comprises a mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Asexplained further below, these amino acid variations are found innaturally-occurring IL-4Ra variants in humans found in many populations.Said human comprises a nucleotide sequence encoding said IL4RA proteincomprising said mutation selected from the group consisting of I75V,E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

A Twenty-Third Configuration provides a ligand (eg, an antibody orantibody fragment) for treating or reducing the risk of anIL4Ra-mediated disease or condition in a human in need thereof, themethod comprising administering to said human said ligand, wherein theligand specifically binds a human IL4RA protein that comprises amutation selected from the group consisting of I75V, E400A, C431R,S503P, Q576R and S752A in SEQ ID NO: 67. Said human comprises anucleotide sequence encoding said IL4RA protein comprising said mutationselected from the group consisting of I75V, E400A, C431R, S503P, Q576Rand S752A in SEQ ID NO: 67.

A Twenty-Fourth Configuration provides a method of targeting IL4Ra in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human IL4RAprotein that comprises a mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said humancomprises a nucleotide sequence encoding said IL4RA protein comprisingsaid mutation selected from the group consisting of I75V, E400A, C431R,S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human issuffering from or at riskof an IL4Ra-mediated disease or condition. Inan example, the method treats or reduces the risk of an IL4Ra-mediateddisease or condition in the human.

A Twenty-Fifth Configuration provides a ligand (eg, an antibody orantibody fragment) for targeting IL4Ra in a human, the method comprisingadministering to said human said ligand, wherein the ligand specificallybinds a human IL4RA protein that comprises a mutation selected from thegroup consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ IDNO: 67. Said human comprises a nucleotide sequence encoding said IL4RAprotein comprising said mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In anexample, the human is suffering from or at riskof an IL4Ra-mediateddisease or condition. In an example, the method treats or reduces therisk of an IL4Ra-mediated disease or condition in the human.

In an embodiment of any of the 21-25^(th) configurations, (i) theantibody or fragment comprises a VH domain derived from therecombination of a human VH segment, a human D gene segment and a humanJH segment, the human VH segment encoding the framework 1 of SEQ ID NO:40 and wherein said human comprises a VH gene segment encoding theframework 1 of SEQ ID NO: 40, or the human expresses VH domains thatcomprise the framework 1 of SEQ ID NO: 40; and wherein (ii) said humancomprises a nucleotide sequence encoding said IL4RA protein comprisingsaid mutation selected from the group consisting of I75V, E400A, C431R,S503P, Q576R and S752A in SEQ ID NO: 67.

Additionally or alternatively, in an embodiment of any of the 21-25^(th)configurations, (i) the antibody or fragment comprises a human gamma-4heavy chain constant region that comprises a Leu at position 189 shownin SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 andwherein said human comprises an IGHG4*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-4 heavy chain constant regions comprising a Leu at position 189shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73;and wherein (ii) said human comprises a nucleotide sequence encodingsaid IL4RA protein comprising said mutation selected from the groupconsisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO:67.

In a Twenty-sixth Configuration, described herein is a method oftreating or reducing the risk of a Nav1.7-mediated disease or conditionin a human in need thereof, the method comprising administering to saidhuman a ligand (eg, an antibody or antibody fragment) that specificallybinds a huma Nav1.7 protein that comprises an amino acid selected fromthe group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693×(wherein X is an amino acid other than E), 767X (wherein X is anamino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G; wherein (i) the ligandcomprises a human gamma-4 heavy chain constant region that comprises aLeu at position 189 shown in SEQ ID NO: 73 or an Arg at position 289shown in SEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01human heavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-4 heavy chain constant regionscomprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises a nucleotide sequence encoding said Nav1.7 protein comprisingsaid amino acid selected from the group consisting of 136I, 216S, 241T,395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T,1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R),328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X isan amino acid other than S), 693 X (wherein X is an amino acid otherthan E), 767X (wherein X is an amino acid other than I), 830X (wherein Xis an amino acid other than R), 897X (wherein X is an amino acid otherthan W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R),1659X (wherein X is an amino acid other than K), 1689X (wherein X is anamino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Theinvention also provides a corresponding ligand, eg, antibody or antibodyfragment, for use in such a method.

In some embodiments, said amino acid is selected from the groupconsisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863Pand 1449V; optionally wherein said disease or condition is a paindisease or condition. In some embodiments, said amino acid is selectedfrom the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V,1464I and 1627K; optionally wherein said disease or condition is a paindisease or condition. In some embodiments, said amino acid is selectedfrom the group consisting of 277X (wherein X is an amino acid other thanR), 328X (wherein X is an amino acid other than Y), 395K, 459X (whereinX is an amino acid other than S), 693 X (wherein X is an amino acidother than E), 767X (wherein X is an amino acid other than I), 830X(wherein X is an amino acid other than R), 897X (wherein X is an aminoacid other than W), 1200L, 1235L, 1488X (wherein X is an amino acidother than R), 1659X (wherein X is an amino acid other than K) and 1689X(wherein X is an amino acid other than W); optionally wherein saiddisease or condition is a pain disease or condition.

In some embodiments, the constant region gene segment comprised by saidhuman is a germline gene segment. In some embodiments, the methodfurther comprises, before said administering, selecting a humancomprising said nucleotide sequence of (ii).

In some embodiments, the human has been determined to comprise thenucleotide sequence that encodes a Nav1.7 protein comprising said aminoacid selected from the group consisting of 136I, 216S, 241T, 395K, 848T,858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,1627K, 277X (wherein X is an amino acid other than R), 328X (wherein Xis an amino acid other than Y), 395K, 459X (wherein X is an amino acidother than S), 693 X (wherein X is an amino acid other than E), 767X(wherein X is an amino acid other than I), 830X (wherein X is an aminoacid other than R), 897X (wherein X is an amino acid other than W),1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X(wherein X is an amino acid other than K), 1689X (wherein X is an aminoacid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G and/or aNav1.7 protein comprising said amino acid selected from the groupconsisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein Xis an amino acid other than R), 328X (wherein X is an amino acid otherthan Y), 395K, 459X (wherein X is an amino acid other than S), 693 X(wherein X is an amino acid other than E), 767X (wherein X is an aminoacid other than I), 830X (wherein X is an amino acid other than R), 897X(wherein X is an amino acid other than W), 1200L, 1235L, 1488X (whereinX is an amino acid other than R), 1659X (wherein X is an amino acidother than K), 1689X (wherein X is an amino acid other than W), 422D,490N, 943L, 1002L, 1161W and 1919G.

In some embodiments, the method further comprises the step ofdetermining that the human comprises (a) the nucleotide sequence thatencodes a Nav1.7 protein comprising said amino acid selected from thegroup consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693 X (wherein X is an amino acid other than E), 767X (wherein X isan amino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G and/or (b) a Nav1.7 proteincomprising said amino acid selected from the group consisting of 136I,216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acidother than R), 328X (wherein X is an amino acid other than Y), 395K,459X (wherein X is an amino acid other than S), 693 X (wherein X is anamino acid other than E), 767X (wherein X is an amino acid other thanI), 830X (wherein X is an amino acid other than R), 897X (wherein X isan amino acid other than W), 1200L, 1235L, 1488X (wherein X is an aminoacid other than R), 1659X (wherein X is an amino acid other than K),1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G, optionally, wherein the determining step isperformed before administration of the antibody to the human. In someembodiments, the step of determining comprises assaying a biologicalsample from the human for a nucleotide sequence encoding a Nav1.7protein comprising said amino acid selected from the group consisting of136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an aminoacid other than R), 328X (wherein X is an amino acid other than Y),395K, 459X (wherein X is an amino acid other than S), 693 X (wherein Xis an amino acid other than E), 767X (wherein X is an amino acid otherthan I), 830X (wherein X is an amino acid other than R), 897X (wherein Xis an amino acid other than W), 1200L, 1235L, 1488X (wherein X is anamino acid other than R), 1659X (wherein X is an amino acid other thanK), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G. In some embodiments, the assaying comprisescontacting the biological sample with at least one oligonucleotide probecomprising a sequence of at least 10 contiguous nucleotides of anucleotide sequence encoding a Nav1.7 protein comprising said amino acidselected from the group consisting of 136I, 216S, 241T, 395K, 848T,858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,1627K, 277X (wherein X is an amino acid other than R), 328X (wherein Xis an amino acid other than Y), 395K, 459X (wherein X is an amino acidother than S), 693 X (wherein X is an amino acid other than E), 767X(wherein X is an amino acid other than I), 830X (wherein X is an aminoacid other than R), 897X (wherein X is an amino acid other than W),1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X(wherein X is an amino acid other than K), 1689X (wherein X is an aminoacid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G orcomprising an antisense sequence of said contiguous nucleotides, whereinsaid sequence of contiguous nucleotides comprises a nucleotide sequenceencoding said amino acid selected from the group consisting of 136I,216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acidother than R), 328X (wherein X is an amino acid other than Y), 395K,459X (wherein X is an amino acid other than S), 693 X (wherein X is anamino acid other than E), 767X (wherein X is an amino acid other thanI), 830X (wherein X is an amino acid other than R), 897X (wherein X isan amino acid other than W), 1200L, 1235L, 1488X (wherein X is an aminoacid other than R), 1659X (wherein X is an amino acid other than K),1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G, thereby forming a complex when at least onenucleotide sequence encoding the Nav1.7 protein comprising said aminoacid selected from the group consisting of 136I, 216S, 241T, 395K, 848T,858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,1627K, 277X (wherein X is an amino acid other than R), 328X (wherein Xis an amino acid other than Y), 395K, 459X (wherein X is an amino acidother than S), 693 X (wherein X is an amino acid other than E), 767X(wherein X is an amino acid other than I), 830X (wherein X is an aminoacid other than R), 897X (wherein X is an amino acid other than W),1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X(wherein X is an amino acid other than K), 1689X (wherein X is an aminoacid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is present;and detecting the presence or absence of the complex, wherein detectingthe presence of the complex determines that the human comprises theNav1.7 protein comprising said amino acid selected from the groupconsisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein Xis an amino acid other than R), 328X (wherein X is an amino acid otherthan Y), 395K, 459X (wherein X is an amino acid other than S), 693 X(wherein X is an amino acid other than E), 767X (wherein X is an aminoacid other than I), 830X (wherein X is an amino acid other than R), 897X(wherein X is an amino acid other than W), 1200L, 1235L, 1488X (whereinX is an amino acid other than R), 1659X (wherein X is an amino acidother than K), 1689X (wherein X is an amino acid other than W), 422D,490N, 943L, 1002L, 1161W and 1919G. In some embodiments, the assayingcomprises nucleic acid amplification and optionally one or more methodsselected from sequencing, next generation sequencing, nucleic acidhybridization, and allele-specific amplification and/or wherein theassaying is performed in a multiplex format. In some embodiments, saidbiological sample comprises serum, blood, faeces, tissue, a cell, urineand/or saliva of said human.

In some embodiments, said human is indicated as heterozygous for anucleotide sequence encoding the Nav1.7 protein comprising said aminoacid selected from the group consisting of 136I, 216S, 241T, 395K, 848T,858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,1627K, 277X (wherein X is an amino acid other than R), 328X (wherein Xis an amino acid other than Y), 395K, 459X (wherein X is an amino acidother than S), 693 X (wherein X is an amino acid other than E), 767X(wherein X is an amino acid other than I), 830X (wherein X is an aminoacid other than R), 897X (wherein X is an amino acid other than W),1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X(wherein X is an amino acid other than K), 1689X (wherein X is an aminoacid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,optionally, wherein said human is further indicated as comprising thenucleotide sequence of SEQ ID NO: 76, or said human is indicated ashomozygous for a nucleotide sequence encoding the Nav1.7 proteincomprising said amino acid selected from the group consisting of 136I,216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D,1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acidother than R), 328X (wherein X is an amino acid other than Y), 395K,459X (wherein X is an amino acid other than S), 693 X (wherein X is anamino acid other than E), 767X (wherein X is an amino acid other thanI), 830X (wherein X is an amino acid other than R), 897X (wherein X isan amino acid other than W), 1200L, 1235L, 1488X (wherein X is an aminoacid other than R), 1659X (wherein X is an amino acid other than K),1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G.

In some embodiments, said human is or has been further determined to besubstantially resistant to a pain or itching treatment. In someembodiments, said human is receiving or has received a pain oranti-itching treatment or has reduced responsiveness to a pain oritching treatment. In some embodiments, said disease or condition is apain or itching disease or condition. In some embodiments, said diseaseor condition is a channelopathy or associated with a channelopathy; oris selected from the group consisting of primary erythermalgia (PE),paroxysmal extreme pain disorder (PEPD) and channelopathy-associatedinsensitivity to pain (CIP). In some embodiments, said human has beendiagnosed with a pain or itching disease or condition.

In some embodiments, said ligand fragment treats or reduces the risk insaid human of a pain or itching disease or condition.

In some embodiments, the nucleotide sequence comprises one or more SNPsselected from the group consisting of rs6746030, rs3750904, rs58022607,rs4369876, rs13402180 and rs12478318.

In a Twenty-seventh Configuration, described herein is a method oftreating or reducing the risk of a disease or condition mediated byVEGF-A in a human, the method comprising administering to said human ananti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibodyfragment) that specifically binds to a human VEGF-A that is expressed bya VEGF-A nucleotide sequence comprising a SNP selected from the groupconsisting of rs699947, rs833061, rs2010963, rs3025039, rs699946,rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360,wherein said human comprises a VEGF-A nucleotide sequence comprisingsaid selected SNP. The invention also provides a corresponding ligand,eg, antibody or antibody fragment, for use in such a method.

In a Twenty-eighth Configuration, described herein is a method oftreating or reducing the risk of a disease or condition mediated byVEGF-A in a human, the method comprising administering to said human ananti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody or antibodyfragment) that specifically binds to a human VEGF-A, wherein the humancomprises (i) an ARMS2 nucleotide sequence comprising a G at theposition of SNP rs10490924; (ii) a CFH nucleotide sequence comprising aTat the position of SNP rs1061170 or an Tat the position of rs3766404;or (iii) a VEGFR2 nucleotide sequence comprising SNP rs4576072 orrs6828477, wherein the ligand comprises a human gamma-1 heavy chainconstant region that comprises an amino acid selected from the groupconsisting of an Asp corresponding to position 204 of SEQ ID NO: 42 anda Leu corresponding to position 206 of SEQ ID NO: 42 and wherein saidhuman comprises an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising said selected amino acid. TheseSNPs are associated with improved responses following anti-VEGF-Atreatment of an ocular disease or condition. The invention also providesa corresponding ligand, eg, antibody or antibody fragment, for use insuch a method.

In a Twenty-ninth Configuration, described herein is a method oftreating or reducing the risk of a VEGF-A mediated disease or conditionin a human, the method comprising administering an anti-human VEGF-Aligand to a human, wherein the human comprises (i) a PDGF-B nucleotidesequence comprising a SNP selected from the group consisting ofrs142404523 (ie, a C corresponding to position −776) and a C at theposition of rs1800818 (ie, a C corresponding to position −735); or (ii)a PDGFR-B nucleotide sequence comprising a SNP selected from the groupconsisting of rs246395 (ie, a G corresponding to position 2601) andrs74943037 (ie, a T corresponding to position 1391). The invention alsoprovides a corresponding ligand, eg, antibody or antibody fragment, foruse in such a method.

In an example of any of the 27-29th configurations, the ligand comprisesan antibody constant region (eg, an antibody Fc region). Optionally, theligand comprises a human gamma-1 heavy chain constant region thatcomprises an amino acid selected from the group consisting of an Aspcorresponding to position 204 of SEQ ID NO: 42 and a Leu correspondingto position 206 of SEQ ID NO: 42 and wherein said human comprises anIGHG1*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-1 heavy chain constantregions comprising said selected amino acid.

In an example of any of the 27-29th configurations the disease orcondition is an ocular condition or a cancer; or angiogenesis orneovascularisation.

In an example of any of the 27-29th configurations the method furthercomprises antagonising PDGF-B in said human.

In an example of any of the 27-29th configurations the method furthercomprises antagonising angiopoietin-2 (Ang2) in said human.

In a Thirtieth configuration, the invention provides:—

A method of treating or reducing the risk of a disease or conditionmediated by PD-L1 in a human, the method comprising administering tosaid human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody orantibody fragment) that specifically binds to a human PD-L1 that isexpressed by a PD-L1 nucleotide sequence comprising a variation selectedfrom the variations listed in Table 21.

For example, there is provided a method of treating or reducing the riskof a disease or condition mediated by PD-L1 in a human, the methodcomprising administering to said human an anti-PD-L1 ligand (eg, ananti-PD-L1 trap, antibody or antibody fragment) that specifically bindsto a human PD-L1 that is expressed by a PD-L1 nucleotide sequencecomprising a variation selected from the variations listed in Table 21,wherein said human comprises a PD-L1 nucleotide sequence comprising saidselected variation.

For example, there is provided a method of treating or reducing the riskof a cancer in a human, the method comprising administering to saidhuman an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibodyfragment) that specifically binds to a human PD-L1 that is expressed bya PD-L1 nucleotide sequence comprising a variation selected from thevariations listed in Table 21, wherein said human comprises a PD-L1nucleotide sequence comprising said selected variation.

For example, there is provided a method of treating or reducing the riskof an autoimmune disease or condition in a human, the method comprisingadministering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1trap, antibody or antibody fragment) that specifically binds to a humanPD-L1 that is expressed by a PD-L1 nucleotide sequence comprising avariation selected from the variations listed in Table 21, wherein saidhuman comprises a PD-L1 nucleotide sequence comprising said selectedvariation.

For example, there is provided a method of treating or reducing the riskinflamatory disease or condition in a human, the method comprisingadministering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1trap, antibody or antibody fragment) that specifically binds to a humanPD-L1 that is expressed by a PD-L1 nucleotide sequence comprising avariation selected from the variations listed in Table 21, wherein saidhuman comprises a PD-L1 nucleotide sequence comprising said selectedvariation.

In a Thirty-first configuration, the invention provides an anti-humanPD-L1 ligand (eg, an antibody, antibody fragment or human PD-L1 trap)for use in a method of the Thirtieth configuration.

In a Thirty-second configuration the invention provides: A method ofcancer immunotherapy in a human by targeting an immune cell TOI (eg,PD-1) in the human, the TOI being present in humans as a plurality ofvariants differing by one or more amino acid polymorphisms, the methodcomprising administering a ligand (eg, an antibody or antibody fragment)to the human, the ligand comprising first and second protein domains,wherein the first domain specifically binds a TOI variant comprising afirst amino acid polymorphism, wherein the second domain comprises asecond polymorphism, and wherein the human expresses (i) TOI comprisingsaid first amino acid polymorphism; and (ii) protein domains comprisingsaid second polymorphism, wherein the ligand comprises a human gamma-4heavy chain constant region that comprises a Leu at position 189 shownin SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 andwherein said human comprises an IGHG4*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-4 heavy chain constant regions comprising a Leu at position 189shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73;and wherein the second domain is comprised by said gamma-4 heavy chainconstant region of the ligand.

This configuration also provides the following aspects:—

A method of cancer immunotherapy in a human by targeting PD-1 in thehuman, the method comprising administering an antibody or antibodyfragment to the human, wherein the antibody or antibody fragmentspecifically binds a PD-1 encoded by a PD-1 nucleotide sequencecomprising a SNP selected from the group consisting of the variationsset out in Table 23, wherein the antibody or antibody fragment comprisesa human gamma-4 heavy chain constant region that comprises a Leu atposition 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown inSEQ ID NO: 73 and wherein said human comprises (i) an IGHG4*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-4 heavy chain constant regionscomprising a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequencecomprising said selected SNP.

A method of cancer immunotherapy in a human by targeting PD-1 in thehuman, the method comprising administering an antibody or antibodyfragment to the human, wherein the antibody or antibody fragmentspecifically binds a PD-1 encoded by a PD-1 nucleotide sequencecomprising a SNP (first polymorphism) selected from the group consistingof the variations set out in Table 23 (eg, selected from the groupconsisting of rs36084323, rs10204225, rs11568821, rs2227981 andrs2227982), wherein the antibody or antibody fragment comprises a humangamma-4 heavy chain constant region that comprises a Leu at position 189shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73and wherein said human comprises (i) an IGHG4*01 human heavy chainconstant region gene segment, or the human expresses antibodiescomprising human gamma-4 heavy chain constant regions comprising a Leuat position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shownin SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence comprising saidselected SNP.

A method of treating cancer in a human, the method comprisingadministering an antibody or antibody fragment to the human, wherein theantibody or antibody fragment inhibits the binding of PD-L1 or PD-L2 toa PD-1 comprising a first amino acid polymorphism, wherein the antibodyor antibody fragment comprises a human gamma-4 heavy chain constantregion that comprises a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73 and wherein said humancomprises (i) an IGHG4*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-4heavy chain constant regions comprising a Leu at position 189 shown inSEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and (ii)a PD-1 nucleotide sequence encoding a PD-1 comprising said first aminoacid polymorphism or the human expresses a PD-1 comprising said firstamino acid polymorphism.

In an alternative, instead of “A method of cancer immunotherapy” or “Amethod of treating cancer”, the invention instead provides “A method oftreating or reducing the risk of an autoimmune disease or condition” or“A method of treating or reducing the risk inflammatory disease orcondition” and the TOI polymorphism is associated with an autoimmune orinflammatory disease or condition.

The invention also provides a ligand (eg, a trap, an antibody orantibody fragment) for use in any of the methods.

In a Thirty-third configuration the invention provides: A method oftreating or reducing the risk of a disease or condition (eg, anaemia) ina human, wherein the disease or condition is mediated by a TOI, the TOIbeing present in humans as a plurality of variants differing by one ormore amino acid polymorphisms, the method comprising administering aligand (eg, an antibody or antibody fragment) to the human, the ligandcomprising first and second protein domains, wherein the first domainspecifically binds a TOI variant comprising a first amino acidpolymorphism, wherein the second domain comprises a second polymorphism,and wherein the human expresses (i) TOI comprising said first amino acidpolymorphism; and (ii) protein domains comprising said secondpolymorphism, optionally wherein the ligand comprises a human gamma-4heavy chain constant region that comprises a Leu at position 189 shownin SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 andwherein said human comprises an IGHG4*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-4 heavy chain constant regions comprising a Leu at position 189shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73;and wherein the second domain is comprised by said gamma-4 heavy chainconstant region of the ligand; wherein, the TOI is selected from thehuman hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.

Examples of any configuration or relating to any TOI are as follows:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4.(vi) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Valat position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shownin SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asnat position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ IDNO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257shown in SEQ ID NO: 6.(vii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(viii) wherein the ligand comprises a human IGLC1*01 lambda chainconstant region and wherein said human comprises (i) a human IGLC1*01lambda chain constant region gene segment, or the human expressesantibodies comprising human IGLC1*01 lambda chain constant regions. (ix)wherein the ligand comprises a human gamma-4 heavy chain constant regionthat comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises (i)an IGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73. (x) wherein the ligandcomprises a human gamma-3 heavy chain constant region encoded by a firsthuman IGHG3 (eg, IGHG3*01) constant region gene segment and wherein saidhuman comprises (i) said first constant region gene segment (eg, anIGHG3*01), or the human expresses antibodies comprising human gamma-3heavy chain constant regions encoded by said first human IGHG3 (eg,IGHG3*01) constant region gene segment.(xi) wherein the ligand comprises a human epsilon heavy chain constantregion encoded by a first human epsilon heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human epsilonheavy chain constant regions encoded by said first constant region genesegment.(xii) wherein the ligand comprises a human mu heavy chain constantregion encoded by a first human mu heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human muheavy chain constant regions encoded by said first constant region genesegment.(xiii) wherein the ligand comprises a human alpha heavy chain constantregion encoded by a first human alpha heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human alphaheavy chain constant regions encoded by said first constant region genesegment.(xiv) wherein the ligand comprises a human delta heavy chain constantregion encoded by a first human delta heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human deltaheavy chain constant regions encoded by said first constant region genesegment.(xv) wherein the ligand comprises a human kappa light chain constantregion encoded by a first human kappa light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human kappalight chain constant regions encoded by said first constant region genesegment.(xvi) wherein the ligand comprises a human lambda light chain constantregion encoded by a first human lambda light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human lambdalight chain constant regions encoded by said first constant region genesegment.

In some embodiments, said ligand (eg, antibody or antibody fragment) isadministered by inhaled, intravenous or subcutaneous administrationand/or is comprised in an inhalable or injectable preparation.

In some embodiments, the human gamma-4 heavy chain constant region ofthe ligand comprises the amino acid sequence of SEQ ID NO: 73 or an ADCCinactivated version thereof.

In some embodiments, the human gamma-4 heavy chain constant regioncomprises 228P and 235E.

Optionally in the present invention the TOI is selected from the groupconsisting of human TOIs: PCSK9, IL6R, IL4Ra, VEGF-A, Placental growthfactor (PGF), PDGF-B, PDGFR-B, Ang-2, Nav1.7, Nav1.8, Nav1.9, PD-1,PD-L1, ICOS, BMP6, hemojuvelin, ferroportin, TMPRSS6, transferrin, humanhemochromatosis protein (HFE) and sclerostin.

For example, the TOI is PCSK9.

For example, the TOI is IL6R.

For example, the TOI is IL4Ra.

For example, the TOI is VEGF-A.

For example, the TOI is Placental growth factor (PGF).

For example, the TOI is PDGF-B.

For example, the TOI is PDGFR-B.

For example, the TOI is Ang-2.

For example, the TOI is Nav1.7.

For example, the TOI is Nav1.8.

For example, the TOI is Nav1.9.

For example, the TOI is PD-1.

For example, the TOI is PD-L1.

For example, the TOI is ICOS.

For example, the TOI is BMP6.

For example, the TOI is hemojuvelin.

For example, the TOI is ferroportin.

For example, the TOI is TMPRSS6.

For example, the TOI is transferrin.

For example, the TOI is human hemochromatosis protein (HFE).

For example, the TOI is sclerostin.

In a Thirty-fourth configuration, described herein is a method oftreating or reducing the risk of a Nav1.8-mediated disease or condition(eg, a pain or cardiac condition) in a human in need thereof, the methodcomprising administering to said human a ligand (eg, an antibody orantibody fragment) that specifically binds a human Nav1.8 protein thatcomprises an amino acid selected from the group consisting of L554P,A1304T, 11706V, A1073V, G1662S, L1092P, I962V, S509P, I206M and R90W oran amino acid encoded by a SNP selected from the group consisting ofrs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,rs74717885 and rs144270136; optionally selected from the groupconsisting of L554P, A1304T, I1706V and G1662S (eg, when the conditionis neuropathic pain); wherein (i) the ligand comprises a human gamma-4heavy chain constant region that comprises a Leu at position 189 shownin SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 andwherein said human comprises (i) an IGHG4*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-4 heavy chain constant regions comprising a Leu at position 189shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73;and wherein (ii) said human comprises a nucleotide sequence encodingsaid Nav1.8 protein comprising said selected amino acid. The inventionalso provides a corresponding ligand, eg, antibody or antibody fragment,for use in such a method.

In alternative embodiments, a VH or VL domain of the ligand (eg,antibody) and/or a different constant region is matched according to theinvention.

In a Thirty-fifth configuration, described herein is a method oftreating or reducing the risk of a Nav1.9-mediated disease or condition(eg, a pain condition) in a human in need thereof, the method comprisingadministering to said human a ligand (eg, an antibody or antibodyfragment) that specifically binds a human Nav1.8 protein that comprisesan amino acid selected from the group consisting of I381T, K419N, A582T,A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091, R86G, T16091and G481E; wherein (i) the ligand comprises a human gamma-4 heavy chainconstant region that comprises a Leu at position 189 shown in SEQ ID NO:73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein saidhuman comprises (i) an IGHG4*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-4heavy chain constant regions comprising a Leu at position 189 shown inSEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; andwherein (ii) said human comprises a nucleotide sequence encoding saidNav1.8 protein comprising said selected amino acid. The invention alsoprovides a corresponding ligand, eg, antibody or antibody fragment, foruse in such a method.

In alternative embodiments, a VH or VL domain of the ligand (eg,antibody) and/or a different constant region is matched according to theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows in silico modeling of PCSK9 surface variant residues.

FIG. 2 depicts the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denoted “C” and the most frequent allele (whichdoes not comprise this SNP) denoted “A”). The figure shows that VH3-23alleles comprising SNP rs56069819 are present at a cumulative frequencyof 11% across all human ethnic populations taken as a whole, whereas incertain specific human ethnic sub-populations (ASW, LWK, YRI, CEU andGBR) such alleles are present at an above-average cumulative frequency.Indicated in the figure are those human PCSK9 variant forms (marked“Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819.

FIG. 3 depicts frameworks and CDRs encoded by VH3-23*04 as obtained fromthe IMGT database (available on the World Wide Web at www.IMGT.org).FIG. 3 discloses the nucleotide sequences as SEQ ID NOS 117, 117, 117,119, 119, 119, 120, 122, 124, 39 and 125, respectively, in order ofappearance. FIG. 3 discloses the coded amino acid sequences as SEQ IDNOS 118, 118, 118, 118, 118, 118, 121, 123, 123, 38 and 126,respectively, in order of appearance.

FIG. 4 depicts sequences of VH3-23*04. The portion of VH3-23*04comprising the FW1 residue change of rs56069819 (SEQ ID NO: 38). Theportion of the nucleic acid sequence encoding rs56069819 is depicted(SEQ ID NO: 39). The FW1 encoded by VH3-23*04 is depicted (SEQ ID NO:40).

DETAILED DESCRIPTION

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in conformation oractivity of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to tailor medicines anddiagnosis of patients more effectively. The present invention providesfor tailored pharmaceuticals and testing that specifically addressesrarer variant forms of a human target of interest (TOD.

The present invention harnesses the power of human genetic variationanalysis and rationally-designed sequence selection. The technicalapplications of these approaches, as per the present invention,contribute to better treatment, prophylaxis and diagnosis in humans andprovides for patient benefit by providing choice and enablingpersonalized medicines and therapies. This provides advantages of betterprescribing, less wastage of medications and improved chances of drugefficacy and better diagnosis in patients.

As sources of genomic sequence variation data, the skilled person willbe aware of the available databases and resources (including updatesthereof) provided by the following:—

-   1. HapMap (The International HapMap Consortium. 2003; available on    the World Wide Web at hapmap.ncbi.nlm.nih.gov/index.html.en). The    HapMap Project is an international project that aims to compare the    genetic sequences of different individuals to identify chromosomal    regions containing shared genetic variants. The HapMap www site    provides tools to identify chromosomal regions and the variant    therein, with options to drill down to population level frequency    data.-   2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010;    available on the World Wide Web at 1000genomes.org/). This resource    provides complete genomic sequence for at least 2500 unidentified    individuals from one of 25 distinct population groups.-   3. Japanese SNP Database (H.Haga et al. 2002; available on the World    Wide Web at snp.ims.u—tokyo.ac.jp/index.html). Based on a study    identifying 190,562 human genetic variants.

The present invention involves the identification and cataloguing ofnaturally-occurring human genomic target sequence variants, includingthose found to be relatively low-frequency or rare variants thatsegregate with specific human ethnic populations and in many individualhumans.

An aspect of the invention is based on rational design of sequenceselection addressing the desirability to tailor medicaments anddiagnostics to rarer, but yet still significant groups of humanindividuals that suffer from, or have the potential to suffer from (ie,who are at risk of), a disease or condition mediated or associated withthe target of interest. In devising this rational design of the presentaspect of the invention, the inventor included considerations of thespread of prevalence of naturally-occurring target variant sequencesacross multiple, diverse human ethnic populations, as well as theimportance of addressing such populations where many individuals arelikely to display a genotype and/or phenotype of one or more of thevariants being analysed. As part of this design, the inventor saw theimportance of adopting the art-recognised classifications of humanethnic populations, and in this respect the inventor based the analysisand design on the recognised human ethnic populations adopted by the1000 Genomes Project, since this is a resource that is, and willcontinue to be, widely adopted by the scientific and medical community.

Thus, in this aspect of the invention, the inventor designed thefollowing variant sequence selection criteria, these being criteria thatthe inventor realised would provide for useful medical drugs anddiagnostics to tailored need in the human population.

Selection Criteria

Three or four of the following:—

-   -   Naturally-occurring human target variant sequences having a        cumulative human allele frequency of 35% or less;    -   Naturally-occurring human target variant sequences having a        total human genotype frequency of 40% or less;    -   Naturally-occurring human target variant sequences found in many        different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project; see Table 4 below);        and    -   Naturally-occurring human target variant sequences found in many        individuals distributed across such many different ethnic        populations.

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingTOI forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20different human ethnic populations (using the standard categorisation ofthe 1000 Genomes Project).

In an embodiment, the naturally-occurring human target variant sequencesare found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individualsdistributed across such many different ethnic populations.

In an example, the following criteria are applied:—

-   -   Naturally-occurring human target variant sequences having a        cumulative human allele frequency of 15% or less;    -   Naturally-occurring human target variant sequences having a        total human genotype frequency of 20% or less;    -   Naturally-occurring human target variant sequences found in at        least 5 different human ethnic populations (using the standard        categorisation of the 1000 Genomes Project); and    -   Naturally-occurring human target variant sequences found in many        individuals distributed across such many different ethnic        populations.

In an example, the criteria are applied with reference to one or morehuman genomic sequence databases as described herein. For example, thecriteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of theinvention, the 1000 Genomes database release 13. For example, the 1000Genomes database in its most recent version as at 1 Oct. 2013.

Optionally, further sequence analysis and 3D in silico modelling (eg,see FIG. 1) can also be used as an additional selection criterion:variants whose variant amino acid residues (versus the most common formof human TOI) are surface-exposed on the target are desirable forselection, since the inventor saw these as contributing to determiningthe topography of the target and potentially contributing to how andwhere ligand binding on the target occurs.

The following bioinformatics protocol is envisaged to identify humansequences for use in the present invention:

-   -   (a) Identify a genomic region containing a target sequence of        interest (target genomic region′) and calculate the genomic        coordinates, using coordinates that match the sequence assembly        build used by either the 1000 Genomes Project or International        HapMap project (or another selected human gene database of        choice).    -   (b) Identify genomic variants mapped to the genomic region        previously identified in (a). Retrieve allele frequencies for        variants for each super population and preferably sub-population        where such data is available. The VWC tools for the 1000 Genomes        Project can be used for this step.    -   (c) Filter list of genomic variants from target genomic region        to contain only variants classed as either ‘non-synonymous’        single nucleotide polymorphisms (SNPs) or genomic ‘insertions or        delections’ (indels). Filter further to include those that are        present in exonic sequences only. “Non-synonymous” refers to        nucleotide variation that produces amino acid variation (ie,        excluding silent mutations).    -   (d) Correlate population frequency data for each of the        identified variants for each of the super populations (for        example ‘European Ancestry’, ‘East Asian ancestry’, ‘West        African ancestry’, ‘Americas’, and ‘South Asian ancestry’) to        identify those variants that segregate with less than two        super-populations. Further correlate all identified variants        with each of the sub-populations (for example, ‘European        ancestry’ super-population might be subdivided into groups such        as ‘CEU—Utah residents with Northern or Western European        ancestry’, ‘TSI Toscani in Italia’ and ‘British from England and        Scotland’) and produce a second score for rarity of variants        within a super-population.    -   (e) Collect one or more sequences that show segregation to        specific sub-populations for use in the present invention, eg,        according to selection criteria as described herein.

Human Populations

Optionally the ethnic populations are selected from those identified inthe 1000 Genomes Project database. In this respect, see Table 4 whichprovides details of the ethnic populations on which the 1000 GenomesProject database is based.

N A Rosenberg et al (Science 20 Dec. 2002: vol. 298 no. 5602 2342-2343)studied the genetic structure of human populations of differinggeographical ancestry. In total, 52 populations were sampled, thesebeing populations with:

African Ancestry

(Mbuti Pygmies, Biaka Pygmies, San peoples, and speakers ofNiger-Kordofanian languages (Bantu, Yoruba or Mandenka populations),

Eurasian Ancestry

(European ancestry (Orcadian, Adygel, Basque, French, Russians,Italians, Sardinian, Tuscan),

Middle Eastern ancestry (Mozabite, Bedouin, Druze, Palestinians),

Central/South Asian ancestry (Balochl, Brahul, Makrani, Sindhi, Pathan,Burusho, Hazara, Uygur, Kalash)),

East Asian Ancestry

(Han, Dal, Daur, Hezhen, Lahu, Miao, Orogen, She, Tujia, Tu, Xibo, Yi,Mongola, Naxi, Cambodian, Japanese, Yakut), Oceanic ancestry(Melanesian, Papuan); or

Americas Ancestry

(Karitiana, Surui, Colombian, Maya, Pima).

The International HapMap Project, Nature, 2003 Dec. 18;426(6968):789-96, discloses that goal of the HapMap Project: todetermine the common patterns of DNA sequence variation in the humangenome by determining the genotypes of one million or more sequencevariants, their frequencies and the degree of association between themin DNA samples from populations with ancestry from parts of Africa, Asiaand Europe. The relevant human populations of differing geographicalancestry include Yoruba, Japanese, Chinese, Northern European andWestern European populations. More specifically:—

Utah population with Northern or Western European ancestry (samplescollected in 1980 by the Centre d'Etude du Polymorphisme Humain (CEPH));

population with ancestry of Yoruba people from Ibadan, Nigeria;

population with Japanese ancestry; and

population with ancestry of Han Chinese from China.

The authors, citing earlier publications, suggest that ancestralgeography is a reasonable basis for sampling human populations.

A suitable sample of human populations used in the present invention isas follows:—

-   -   (a) European Ancestry    -   (b) Northern European ancestry; Western European ancestry;        Toscani ancestry; British ancestry, Finnish ancestry or Iberian        ancestry.    -   (c) More specifically, population of Utah residents with        Northern and/or Western European ancestry; Toscani population in        Italia; British population in England and/or Scotland; Finnish        population in Finland; or Iberian population in Spain.    -   (a) East Asian ancestry    -   (b) Japanese ancestry; Chinese ancestry or Vietnamese ancestry.    -   (c) More specifically, Japanese population in Toyko, Japan; Han        Chinese population in Beijing, China; Chinese Dai population in        Xishuangbanna; Kinh population in Ho Chi Minh City, Vietnam; or        Chinese population in Denver, Colo., USA.    -   (a) West African ancestry    -   (b) Yoruba ancestry; Luhya ancestry; Gambian ancestry; or        Malawian ancestry.    -   (c) More specifically, Yoruba population in Ibadan, Nigeria;        Luhya population in Webuye, Kenya; Gambian population in Western        Division, The Gambia; or Malawian population in Blantyre,        Malawi.    -   (a) Population of The Americas    -   (b) Native American ancestry; Afro-Caribbean ancestry; Mexican        ancestry; Puerto Rican ancestry; Columbian ancestry; or Peruvian        ancestry.    -   (c) More specifically, population of African Ancestry in        Southwest US; population of African American in Jackson, Miss.;        population of African Caribbean in Barbados; population of        Mexican Ancestry in Los Angeles, Calif.; population of Puerto        Rican in Puerto Rico; population of Colombian in Medellin,        Colombia; or population of Peruvian in Lima, Peru.    -   (a) South Asian ancestry    -   (b) Ahom ancestry; Kayadtha ancestry; Reddy ancestry; Maratha;        or Punjabi ancestry.    -   (c) More specifically, Ahom population in the State of Assam,        India; Kayadtha population in

Calcutta, India; Reddy population in Hyderabad, India; Marathapopulation in Bombay, India; or Punjabi population in Lahore, Pakistan.

In any configuration of the invention, in one embodiment, each humanpopulation is selected from a population marked “(a)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(b)” above.

In any configuration of the invention, in another embodiment, each humanpopulation is selected from a population marked “(c)” above.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with European ancestry, an ethnicpopulation with East Asian, an ethnic population with West Africanancestry, an ethnic population with Americas ancestry and an ethnicpopulation with South Asian ancestry.

In one embodiment the ethnic populations are selected from the groupconsisting of an ethnic population with Northern European ancestry; oran ethnic population with Western European ancestry; or an ethnicpopulation with Toscani ancestry; or an ethnic population with Britishancestry; or an ethnic population with Icelandic ancestry; or an ethnicpopulation with Finnish ancestry; or an ethnic population with Iberianancestry; or an ethnic population with Japanese ancestry; or an ethnicpopulation with Chinese ancestry; or an ethnic population Vietnameseancestry; or an ethnic population with Yoruba ancestry; or an ethnicpopulation with Luhya ancestry; or an ethnic population with Gambianancestry; or an ethnic population with Malawian ancestry; or an ethnicpopulation with Native American ancestry; or an ethnic population withAfro-Caribbean ancestry; or an ethnic population with Mexican ancestry;or an ethnic population with Puerto Rican ancestry; or an ethnicpopulation with Columbian ancestry; or an ethnic population withPeruvian ancestry; or an ethnic population with Ahom ancestry; or anethnic population with Kayadtha ancestry; or an ethnic population withReddy ancestry; or an ethnic population with Maratha; or an ethnicpopulation with Punjabi ancestry.

Anti-Target Ligands

The invention provides useful anti-target ligands for addressing humanssuffering from or likely to suffer from a disease or condition mediatedor associated with the TOI. For example, the ligand specifically bindsto the TOI variant as per the invention. The ligand may inhibit orantagonise the activity of the target, eg, the ligand neutralises thetarget. The skilled person will be familiar with neutralising ligands ingeneral, such as antibodies or antibody fragments, and can readily testsuitable ligands for specific binding and/or neutralisation of a targetin vitro or in an in vivo assay.

An antibody “fragment” comprises a portion intact antibody, preferablythe antigen binding and/or the variable region of the intact antibody.Examples of antibody fragments include dAb, Fab, Fab′, F(ab′)2 and Fvfragments; diabodies; linear antibodies; single-chain antibody moleculesand multispecific antibodies formed from antibody fragments.

In an embodiment, the ligand of the invention is or comprises anantibody or antibody fragment, for example an antibody or fragmentcomprising human variable regions (and optionally also human constantregions). Anti-TOI or TOI-binding or targeting antibodies and fragmentscan be prepared according to any known method, eg, using transgenic mice(eg, the Kymouse™ or Velocimouse™, or Omnimouse™, Xenomouse™, HuMabMouse™ or MeMo Mouse™), rats (eg, the Omnirat™), camelids, sharks,rabbits, chickens or other non-human animals immunised with the TOIfollowed optionally by humanisation of the constant regions and/orvariable regions to produce human or humanised antibodies. In anexample, display technologies can be used, such as yeast, phage orribosome display, as will be apparent to the skilled person. Standardaffinity maturation, eg, using a display technology, can be performed ina further step after isolation of an antibody lead from a transgenicanimal, phage display library or other library. Representative examplesof suitable technologies are described in US20120093818 (Amgen, Inc),which is incorporated herein by reference, eg, the methods set out inparagraphs [0309] to [0346]. Although this is with reference to PCSK9,the antibody-generating methods can be applied to other TOIs as per thebroadest scopes of the present invention.

Generally, a VELOCIMMUNE™ or other mouse or rat can be challenged withthe antigen of interest, and lymphatic cells (such as B-cells) arerecovered from the mice that express antibodies. The lymphatic cells maybe fused with a myeloma cell line to prepare immortal hybridoma celllines, and such hybridoma cell lines are screened and selected toidentify hybridoma cell lines that produce antibodies specific to theantigen of interest. DNA encoding the variable regions of the heavychain and light chain may be isolated and linked to desirable isotypicconstant regions of the heavy chain and light chain. Such an antibodyprotein may be produced in a cell, such as a CHO cell. Alternatively,DNA encoding the antigen-specific chimaeric antibodies or the variabledomains of the light and heavy chains may be isolated directly fromantigen-specific lymphocytes.

Initially, high affinity chimaeric antibodies are isolated having ahuman variable region and a mouse constant region. As described below,the antibodies are characterized and selected for desirablecharacteristics, including affinity, selectivity, epitope, etc. Themouse constant regions are replaced with a desired human constant regionto generate the fully human antibody of the invention, for examplewild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 751, 752,753in US2011/0065902, which sequences are incorporated herein by referencefor use in the ligands of the present invention). While the constantregion selected may vary according to specific use, high affinityantigen-binding and target specificity characteristics reside in thevariable region.

In an example, the ligand of the invention is or comprises a nucleicacid, eg, RNA, eg, siRNA that hybridises under stringent condition tothe TOI variant sequence, eg, hybridises a nucleotide sequencecomprising one or more nucleotides that are variant (versus the mostcommon TOI sequence, eg, with reference to the 1000 Genomes Projectdatabase).

Target binding ability, specificity and affinity (K_(d), K_(off) and/orK_(on)) can be determined by any routine method in the art, eg, bysurface plasmon resonance (SPR). The term “Kd”, as used herein, isintended to refer to the equilibrium dissociation constant of aparticular antibody-antigen interaction.

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0 05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand (eg, antibody) is determinedusing SPR by

-   1. Coupling anti-mouse (or other relevant human, rat or non-human    vertebrate antibody constant region species-matched) IgG (eg,    Biacore™ BR-1008-38) to a biosensor chip (eg, GLM chip) such as by    primary amine coupling;-   2. Exposing the anti-mouse IgG (or other matched species antibody)    to a test IgG antibody to capture test antibody on the chip;-   3. Passing the test antigen over the chip's capture surface at 1024    nM, 256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and-   4. And determining the affinity of binding of test antibody to test    antigen using surface plasmon resonance, eg, under an SPR condition    discussed above (eg, at 25° C. in physiological buffer). SPR can be    carried out using any standard SPR apparatus, such as by Biacore™ or    using the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an example, the ligand of the invention is contained in a medicalcontainer, eg, a vial, syringe, IV container or an injection device (eg,an intraocular or intravitreal injection device). In an example, theligand is in vitro, eg, in a sterile container. In an example, theinvention provides a kit comprising the ligand of the invention,packaging and instructions for use in treating or preventing ordiagnosing in a human a disease or condition mediated by the TOI. In anexample, the instructions indicate that the human should be genotypedfor a TOI variant sequence of the invention before administering theligand to the human. In an example, the instructions indicate that thehuman should be phenotyped for a TOI variant of the invention beforeadministering the ligand to the human. In an example, the human is ofChinese (eg, Han) ethnicity and the instructions are in Chinese (eg,Mandarin). In an example, the instructions comprise directions toadminister alirocumab or evolocumab to said human.

The invention relates to the concepts set out in the following clauses.

Clause 1 A method of treating or preventing a disease or condition in ahuman, wherein the disease or condition is mediated by a Target ofInterest (TOI), wherein the TOI is present in humans as differentpolymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is encoded by a nucleotide sequence having        a cumulative human allele frequency of less than 50% and/or        wherein the TOI in said human is encoded by a nucleotide        sequence having a total human genotype frequency of less than        50%;    -   wherein    -   Before step (a) said human has been or is genotyped as positive        for said nucleotide sequence or phenotyped as positive for said        TOI variant, or the method comprises before step (a) genotyping        the human as positive for said nucleotide sequence or        phenotyping the human as positive for said TOI variant.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined using bioinformatics.

In any aspect, configuration, example, embodiment, clause or conceptherein, frequencies may be determined by reference to a databasecomprising at least 1000 or 2000 human sequences.

In any aspect, configuration, example, embodiment, clause or conceptherein “heterozygous human genotype frequency” means the cumulativefrequency of all genotypes in the sample or database or in humans havingone occurrence of the rare variant allele and one occurrence of anotherallele (heterozygous state), eg, genotype in 1000 Genomes database.

In any aspect, configuration, example, embodiment, clause or conceptherein “homozygous human genotype frequency means the cumulativefrequency of two occurrences of the variant allele (homozygous state),eg, genotype in 1000 Genomes Project database.

In any aspect, configuration, example, embodiment, clause or conceptherein “total human genotype frequency” means the total of heterozygousplus homozygous human genotype frequencies.

In any aspect, configuration, example, embodiment, clause or conceptherein “cumulative human allele frequency” refers to the total of alloccurrences of the variant allele (eg, SNP) in the sample or database orin humans, eg, in the 1000 Genomes Project database.

Clause 2: The method of clause 1, wherein before step (a) the ligand hasbeen or is determined as being capable of binding to said TOI variant,eg, with an affinity (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliserof the TOI. In an example, determination is carried out in a human (eg,in a clinical trial). In an example, determination is carried out in anon-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-humanprimate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 3: A method of treating or preventing a disease or condition in ahuman, wherein the disease or condition is mediated by a Target ofInterest (TOI), wherein the TOI is present in humans as differentpolymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is encoded by a nucleotide sequence having        a cumulative human allele frequency of less than 50% and/or        wherein the TOI in said human is encoded by a nucleotide        sequence having a total human genotype frequency of less than        50%;        -   wherein    -   b. Before step (a) the ligand has been or is determined as        capable of binding to said TOI variant, eg, with an affinity        (Kd) disclosed below.

In an example, the ligand is (or has been determined as) a neutraliserof the TOI. In an example, determination is carried out in a human (eg,in a clinical trial). In an example, determination is carried out in anon-human, eg, in a mouse, rat, rabbit, pig, dog, sheep or non-humanprimate (eg, Cynomolgous monkey, rhesus monkey or baboon).

Clause 4: The method of clause 3, wherein the genome of said humancomprises a nucleotide sequence encoding said TOI variant; and beforestep (a) said nucleotide sequence has been or is determined as having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%.

The TOI variant is not the most frequent.

Clause 5: The method of clause 3 or 4, wherein said human has been or isgenotyped as positive for said variant nucleotide sequence before step(a), or the method comprises genotyping the human as positive for saidvariant nucleotide sequence before step (a).

Clause 6: The method of any preceding clause, wherein the human has beenor is phenotyped as positive for said TOI variant before step (a), orthe method comprises phenotyping the human as positive for said variantnucleotide sequence before step (a).

Clause 7: The method of any preceding clause, wherein said frequency isless than 10 or 15% (eg, from 1 to 10%).

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

Clause 8: The method of any preceding clause, wherein the ligand iscapable of binding to two or more different TOI variants, each beingencoded by a nucleotide sequence having a cumulative human allelefrequency of less than 50% (eg, from 1 to 10%) and/or having a totalhuman genotype frequency of less than 50% (eg, from 1 to 20%).

In an embodiment, the cumulative human allele frequency of each TOIvariant is 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to20% or 1 to 15% or 1 to 10%.

In an embodiment, the total human genotype frequency of each TOI variantis 35, 30, 25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%,1 to 20%, 1 to 15%, 1 to about 15%, 1 to 10%, I to about 10% or 1 to 5%or 1 to about 5%.

Clause 9: A method of treating or preventing a disease or condition in ahuman, wherein the disease or condition is mediated by a Target ofInterest (TOI), wherein the TOI is present in humans as differentpolymorphic variants, the method comprising

-   -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent (eg, by at least 40,        50, 60, 70, 80, 90 or 95%) said disease or condition, wherein        the TOI in said human is a variant encoded by a nucleotide        sequence having a cumulative human allele frequency of more than        50% (eg, the highest frequency) and/or having a total human        genotype frequency of more than 50% (eg, the highest frequency);    -   wherein    -   b. Before step (a) said human has been or is genotyped as        negative for a variant nucleotide sequence having a cumulative        human allele frequency of less than 50% and/or having a total        human genotype frequency of less than 50%; or phenotyped as        negative for a TOI variant encoded by a nucleotide sequence        having a cumulative human allele frequency of less than 50%        and/or having a total human genotype frequency of less than 50%;    -   or    -   Before step (a) said the method comprises genotyping the human        as negative for a variant nucleotide sequence having a        cumulative human allele frequency of less than 50% and/or having        a total human genotype frequency of less than 50%; or        phenotyping the human as negative for a TOI variant encoded by a        nucleotide sequence having a cumulative human allele frequency        of less than 50% and/or having a total human genotype frequency        of less than 50%.

In an embodiment, in (a) the cumulative human allele frequency is 55,60, 65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or100% (eg, in the range from 51 to 80%).

In an embodiment, in (a) the total human genotype frequency is 55, 60,65, 70, 75, 80, 85 or 90 or more but less than 95, 96, 97, 98, 99 or100% (eg, in the range from 51 to 80%).

In an embodiment, in (b) the cumulative human allele frequency is 30,25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15%or 1 to 10%.

In an embodiment, in (b) the total human genotype frequency is 35, 30,25, 20, 15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%,1 to 15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 toabout 5%.

Clause 10: The method of clause 9, wherein before step (a), the humanhas been or is phenotyped as positive for the most frequent TOI variantor genotyped for the nucleotide sequence thereof

In an embodiment, before step (a) the human has been or is genotyped aspositive for TOI variant nucleotide sequence having a cumulative humanallele frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but lessthan 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) orphenotyped for the TOI variant thereof.

In an embodiment, before step (a) the human has been or is genotyped aspositive for TOI variant nucleotide sequence having a total humangenotype frequency of 55, 60, 65, 70, 75, 80, 85 or 90 or more but lessthan 95, 96, 97, 98, 99 or 100% (eg, in the range from 51 to 80%) orphenotyped for the TOI variant thereof.

Clause 11: The method of clause 9 or 10, wherein before step (a) theligand has been or is determined as being capable of binding to the mostfrequent TOI variant.

Clause 12: The method of clause 9, 10 or 11, wherein before step (a) theligand has been or is determined as being substantially incapable ofneutralising or inhibiting said TOI variant recited in step (b).

By “substantially incapable or neutralising or inhibiting” is meant:Neutralisation or inhibition less than 50, 25, 10, 5 or 0.5% inhibitionor neutralisation of the most frequent TOI variant.

Clause 13: The method of any one of clauses 9 to 12, wherein the ligandis capable of binding to the most frequent TOI variant.

Clause 14: The method of any one of clauses 9 to 13, wherein the ligandis capable of binding to two or more different TOI variants, each beingencoded by a nucleotide sequence having a cumulative human allelefrequency of more than 50%.

In an embodiment, each TOI variant is encoded by a nucleotide sequencehaving a cumulative human allele frequency of 55, 60, 65, 70, 75, 80, 85or 90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the rangefrom 51 to 80%).

In an embodiment, each TOI variant is encoded by a nucleotide sequencehaving a total human genotype frequency of 55, 60, 65, 70, 75, 80, 85 or90 or more but less than 95, 96, 97, 98, 99 or 100% (eg, in the rangefrom 51 to 80%).

Clause 15: The method of any preceding clause, wherein said variantnucleotide sequence recited in step (a) has been or is determined asbeing present in at least 2 different human ethnic populations, eg, atleast 2, 3, 4, 5, 6, 7, 8 or 9 different human ethnic populations inTable 4.

Clause 16: The method of any preceding clause, wherein said humanfrequency is the frequency in a database of naturally-occurringsequences sampled from at least 15, 20 or 25 different human ethnicpopulations and comprising at least 1000 sequences. In an embodiment,the database is the 1000 Genomes Project database as described herein.

Clause 17: An anti-human TOI ligand for use in a method of treatingand/or preventing a TOI-mediated disease or condition in a human,wherein the TOI is present in humans as different polymorphic variantsand wherein the genome of said human comprises a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, the method comprisingadministering the ligand to the human.

In the alternative, clause 17 provides an anti-human TOI ligand for usein a method according to any one of clauses 1 to 16, the methodcomprising administering the ligand to the human.

In an embodiment, the cumulative human allele frequency is 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 20% or 1 to 15% or 1 to10%.

In an embodiment, the total human genotype frequency is 35, 30, 25, 20,15, 10 or 5% or less, eg, in the range from 1 to 25%, 1 to 20%, 1 to15%, 1 to about 15%, 1 to 10%, 1 to about 10% or 1 to 5% or 1 to about5%.

Clause 18: The ligand of clause 17, wherein the ligand has been or isdetermined as capable of binding the human TOI encoded by saidnucleotide sequence.

In the alternative, clause 18 provides a ligand that binds a human TOIcomprising an amino acid sequence encoded by a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, for use in a methodcomprising the step of using the ligand to target said TOI in a human totreat and/or prevent a disease or condition mediated by TOI, the methodcomprising administering the ligand to the human.

Clause 19: A ligand that binds a human TOI comprising an amino acidsequence encoded by a TOI nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, for use in a method according to any one ofclauses 1 to 16, the method comprising administering the ligand to thehuman.

Clause 20: The ligand of any one of clauses 17 to 19, wherein the humanhas been or is genotyped as positive for said TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50%.

The ligand of any one of clauses 17 to 19, wherein the human has been oris genotyped as positive for said TOI nucleotide sequence having a totalhuman genotype frequency of less than 50%.

Clause 21: The ligand of any one of clauses 17 to 20, wherein the humanhas been or is phenotyped as positive for a TOI encoded by a nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

Clause 22: The ligand of any one of clauses 17 to 21, wherein the humanhas been or is genotyped as heterozygous for a TOI nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/orhaving a total human genotype frequency of less than 50%; optionallywherein the human has been or is genotyped as comprising a TOInucleotide sequence having a cumulative human allele frequency of lessthan 50% and a TOI nucleotide sequence having a cumulative human allelefrequency of more than 50% (eg, having the highest cumulative humanallele frequency) and/or having a total human genotype frequency of morethan 50% (eg, having the highest total human genotype frequency).

Clause 23: The ligand of any one of clauses 17 to 22, wherein the genomeof the human has been or is genotyped as homozygous for a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or having a total human genotype frequency of less than 50%.

Clause 24: The ligand of any one of clauses 17 to 23, wherein the ligandcomprises an antibody binding site that binds a human TOI comprising anamino acid sequence encoded by a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%; and optionally has been or isdetermined as capable of such binding.

Clause 25: The ligand of clause 24, wherein the ligand is an antibody orantibody fragment.

Clause 26:The ligand of any one of clauses 17 to 23, wherein the ligandcomprises a nucleotide sequence that specifically hybridises to a TOInucleotide sequence having a cumulative human allele frequency of lessthan 50% and/or having a total human genotype frequency of less than 50%or an RNA transcript thereof; and/or the ligand comprises a nucleotidesequence that comprises at least 10 contiguous nucleotides of anucleotide sequence having a cumulative human allele frequency of lessthan 50% and/or having a total human genotype frequency of less than 50%or is an antisense sequence thereof

In an embodiment, the ligand comprises a nucleotide sequence thatcomprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or100 contiguous nucleotides of a nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or having a total humangenotype frequency of less than 50% or is an antisense sequence thereof.

Clause 27: The ligand of any one of clauses 17 to 26, wherein the genomeof said human comprises a nucleotide sequence having a cumulative humanallele frequency of less than 50% and the sequence is found in at least2 different ethnic populations (eg, found in at least 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (forexample as per the populations in Table 4)). In an example, numbers arewith reference to the 1000 Genomes Project database.

The ligand of any one of clauses 17 to 26, wherein the genome of saidhuman comprises a nucleotide sequence having a cumulative human allelefrequency of less than 50% and the sequence is found in at least 20individuals distributed across at least 2 said different ethnicpopulations (eg, found in at least in at least 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130,140 or 150 individuals distributed across such many different ethnicpopulations). In an example, numbers are with reference to the 1000Genomes Project database.

Clause 28: A pharmaceutical composition or kit for treating orpreventing a condition or disease mediated by a TOI as recited in anypreceding clause, the composition or kit comprising a ligand of any oneof clauses 17 to 27; and optionally in combination with a label orinstructions for use to treat and/or prevent said disease or conditionin a human; optionally wherein the label or instructions comprise amarketing authorisation number (eg, an FDA or EMA authorisation number);optionally wherein the kit comprises an injection pen or IV containerthat comprises the ligand.

In an example, the label or instructions cover or describe use for ahuman comprising a TOI variant encoded by a nucleotide sequence asrecited in clause 17.

Clause 29: A method of producing an anti-human TOI antibody bindingsite, the method comprising obtaining a plurality of anti-TOI antibodybinding sites, screening the antibody binding sites for binding to a TOIcomprising an amino acid sequence encoded by a nucleotide sequencehaving a cumulative human allele frequency of less than 50% and/or atotal human genotype frequency of less than 50%, or to a peptide thereofthat comprises an amino acid variation from the corresponding sequenceencoded by the TOI-encoding nucleotide sequence having the highestcumulative human allele frequency and/or the highest total humangenotype frequency, and isolating an antibody binding site that binds inthe screening step.

In an embodiment of any aspect herein, the antibody, fragment or bindingsite is recombinant

In the alternative, clause 29 provides: A method of producing ananti-human TOI antibody, the method comprising immunising a non-humanvertebrate (eg, a mouse or a rat) with a TOI comprising an amino acidsequence encoded by a nucleotide sequence having a cumulative humanallele frequency of less than 50% and/or a total human genotypefrequency of less than 50%, or to a peptide thereof that comprises anamino acid variation from the corresponding sequence encoded by theTOI-encoding nucleotide sequence having the highest cumulative humanallele frequency and/or the highest total human genotype frequency, andisolating an antibody that binds a TOI comprising an amino acid sequenceencoded by a TOI nucleotide sequence having a cumulative human allelefrequency of less than 50% and/or a total human genotype frequency ofless than 50%, and optionally producing a TOI-binding fragment orderivative of the isolated antibody.

The term “isolated” with reference to a ligand, antibody or protein, forexample in any aspect, configuration, example or embodiment, means thata subject ligand, antibody, protein etc (1) is free of at least someother proteins with which it would normally be found, (2) is essentiallyfree of other proteins from the same source, e.g., from the samespecies, (3) is expressed by a cell from a different species, (4) hasbeen separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is associated innature, (5) is operably associated (by covalent or noncovalentinteraction) with a polypeptide with which it is not associated innature, or (6) does not occur in nature. Typically, an “isolated”ligand, antibody, protein etc constitutes at least about 5%, at leastabout 10%, at least about 25%, or at least about 50% of a given sample.Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or anycombination thereof can encode such an isolated ligand, antibody proteinetc. Preferably, the isolated ligand, antibody protein etc issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

For example, an “isolated” antibody is one that has been identified,separated and/or recovered from a component of its productionenvironment (eg, naturally or recombinantly). Preferably, the isolatedpolypeptide is free of association with all other components from itsproduction environment, eg, so that the antibody has been isolated to anFDA-approvable or approved standard. Contaminant components of itsproduction environment, such as that resulting from recombinanttransfected cells, are materials that would typically interfere withresearch, diagnostic or therapeutic uses for the antibody, and mayinclude enzymes, hormones, and other proteinaceous or non-proteinaceoussolutes. In preferred embodiments, the polypeptide will be purified: (1)to greater than 95% by weight of antibody as determined by, for example,the Lowry method, and in some embodiments, to greater than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes the antibody in situ within recombinant cellssince at least one component of the antibody's natural environment willnot be present. Ordinarily, however, an isolated polypeptide or antibodywill be prepared by at least one purification step.

Immunoconjugates

The invention encompasses the ligand (eg, antibody) conjugated to atherapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxinagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxin agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, see for example, WO 05/103081.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-TOI (eg, anti-PCSK9)mAbs can be linked to or co-expressed with another functional molecule,e.g., another peptide or protein. For example, an antibody or fragmentthereof can be functionally linked (e.g., by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody or antibody fragment, toproduce a bispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Clause 30: The method of clause 29, comprising the step of obtaining anucleic acid encoding the antibody, fragment, derivative or binding siteand optionally inserting the nucleic acid in an expression vector.

Clause 31: A kit for TOI genotyping a human, wherein the kit comprises anucleic acid comprising a nucleotide sequence that specificallyhybridises to a TOI nucleotide sequence selected having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50% or an RNA transcript thereof and/or thenucleic acid comprises a nucleotide sequence that comprises at least 10(eg, at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100)contiguous nucleotides of a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or a total human genotypefrequency of less than 50% or is an antisense sequence thereof.

For example, the nucleic acid hybridises to a region immediatelyflanking a nucleotide that is variant compared to the correspondingnucleotide of the TOI nucleotide sequence having the highest cumulativehuman allele frequency and/or the highest total human genotypefrequency. In an example, the nucleic acid hybridises to at two or moresuch variant nucleotides.

Specific hybridisation is under stringent conditions, as will beapparent to the skilled person, eg, conditions of 5×SSC, 5×Denhardt'sreagent, and 0.5% SDS at 65° C.

Clause 32: A kit for TOI genotyping or phenotyping a human, wherein thekit comprises a ligand according to any one of clauses 17 to 27 or anantibody, fragment or derivative produced by the method of any one ofclauses 29 to 31.

For example, the ligand specifically binds to an epitope comprising anamino acid that is variant compared to the corresponding amino acid ofthe TOI encoded by a nucleotide sequence having the highest cumulativehuman allele frequency and/or the highest total human genotypefrequency. In an example, the ligand specifically binds to an epitopecomprising two or more such variant amino acids. In an example, specificbinding means binding with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1nM or less, eg, as determined by SPR.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

Clause 33: Use of an anti-TOI ligand that binds a human TOI comprisingan amino acid sequence encoded by a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%, in the manufacture of a medicamentfor treating and/or preventing a TOI-mediated disease or condition in ahuman whose genome comprises a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or having a totalhuman genotype frequency of less than 50%.

Clause 34: Use of an anti-TOI ligand that binds a human TOI comprisingan amino acid sequence encoded by a TOI nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%, in the manufacture of a medicamentfor targeting said TOI in a human to treat and/or prevent a disease orcondition mediated by TOI.

The use of clause 33 or 34, wherein the ligand, human, disease orcondition is according to any one of clauses 1 to 27.

Clause 35: A method of targeting a TOI for treating and/or preventing aTOI-mediated disease or condition in a human, the method comprisingadministering an anti-TOI ligand to a human comprising a TOI nucleotidesequence selected having a cumulative human allele frequency of lessthan 50% and/or a total human genotype frequency of less than 50%,whereby a TOI encoded by said nucleotide sequence is targeted.

Clause 36: The method of clause 35, wherein the method comprisestargeting a human TOI comprising an amino acid sequence with said ligandto treat and/or prevent said disease or condition in said human, whereinsaid amino acid sequence is encoded by a nucleotide sequence having acumulative human allele frequency of less than 50% and/or a total humangenotype frequency of less than 50%.

Clause 37: A method of TOI genotyping a nucleic acid sample of a human,the method comprising identifying in the sample the presence of a TOInucleotide sequence having a cumulative human allele frequency of lessthan 50% and/or having a total human genotype frequency of less than50%.

In an example, the method comprises obtaining a TOI nucleic acid samplefrom the human and then carrying out the identifying step.

Clause 38: A method of TOI typing a protein sample of a human, themethod comprising identifying in the sample the presence of a TOI aminoacid sequence encoded by a TOI nucleotide sequence having a cumulativehuman allele frequency of less than 50% and/or having a total humangenotype frequency of less than 50%.

In an example, the method comprises obtaining a TOI protein sample fromthe human and then carrying out the identifying step.

Clause 39: The method of clause 37 or 38, comprising obtaining a sampleof serum, blood, faeces, hair, urine or saliva from a human, whereby thenucleic acid or protein sample is obtained for use in the step ofidentifying said sequence.

Clause 40: The method of any one of clauses 37 to 39, comprising using aligand according to any one of clauses 17 to 27 to carry out saididentifying step.

Clause 41: A diagnostic kit comprising a ligand that is capable ofbinding a human TOI comprising an amino acid sequence encoded by a TOInucleotide sequence having a cumulative human allele frequency of lessthan 50% and/or a total human genotype frequency of less than 50% andinstructions for carrying out the method of clause 38 or 39.

Clause 42: A diagnostic kit comprising a nucleic acid probe comprising anucleotide sequence that specifically hybridises a TOI nucleotidesequence having a cumulative human allele frequency of less than 50%and/or a total human genotype frequency of less than 50% or an RNAtranscript thereof and instructions for carrying out the method ofclause 38 or 39.

Clause 43: The method, ligand, composition, kit or use of any precedingclause, wherein the TOI is encoded by a nucleotide sequence having acumulative human allele frequency from 1 to 10% and/or a total humangenotype frequency from 1 to about 15% or from 1 to 15%.

Clause 44: The method, ligand, composition, kit or use of any precedingclause wherein the TOI is a human TOI selected from Table 5; optionallyfor treating and/or preventing a corresponding disease or condition asset out in Table 5.

For example, the TOI is human PCSK9, eg, a mature, cleaved,autocatalysed or active PCSK9. In an example, the disease is acardiovascular disease such as hyperlipidaemia.

Ligands of the invention are useful, for instance, in specific bindingassays, for genotyping or phenotyping humans, affinity purification ofthe TOI and in screening assays to identify other antagonists of TOIactivity. Some of the ligands of the invention are useful for inhibitingbinding of TOI to a congnate human receptor or protein, or inhibitingTOI-mediated activities.

The invention encompasses anti-TOI (eg, PCSK9) antibody ligands having amodified glycosylation pattern. In some applications, modification toremove undesirable glycosylation sites may be useful, or e.g., removalof a fucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand is or comprisesa recombinant human antibody or fragment thereof which specificallybinds the TOI (eg, a rare variant as described herein) and apharmaceutically acceptable carrier. In one embodiment, the inventionfeatures a composition which is a combination of an antibody ligand orantigen-binding fragment of an antibody of the invention, and a secondtherapeutic agent. The second therapeutic agent may be any of ananti-inflammatory agent, an anti-angiogenesis agent, a painkiller, adiuretic, a chemotherapeutic agent, an anti-neoplastic agent, avasodilator, a vasoconstrictor, a statin, a beta blocker, a nutrient, anadjuvant, an anti-obesity agent and an anti-diabetes agent.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the USA Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans. A “pharmaceutically acceptable carrier,excipient, or adjuvant” refers to an carrier, excipient, or adjuvantthat can be administered to a subject, together with an agent, e.g., anyantibody or antibody chain described herein, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the agent.

In an example, the invention features a method for inhibiting TOIactivity using the anti-TOI ligand of the invention (eg, an antibody orantigen-binding portion of the antibody of the invention), wherein thetherapeutic method comprises administering a therapeutically effectiveamount of a pharmaceutical composition comprising the ligand. Thedisorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof TOI activity.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The term “specifically binds,” or the like, means that a ligand, eg, anantibody or antigen-binding fragment thereof, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁶M or less (e.g., a smaller KD denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds a human TOI may, however, exhibitcross-reactivity to other antigens such as a TOI molecule from anotherspecies. Moreover, multi-specific antibodies (e.g., bispecifics) thatbind to human TOI and one or more additional antigens are nonethelessconsidered antibodies that “specifically bind” TOI, as used herein.

Genotyping & Phenotyping

The skilled person will be familiar with techniques that can be used foraccurate genotyping and application to the invention. These include thefollowing.

1 Hybridization-based methods

1.1 Dynamic allele-specific hybridization

1.2 Molecular beacons

1.3 SNP microarrays

2 Enzyme-based methods

2.1 Restriction fragment length polymorphism

2.2 PCR-based methods

2.3 Flap endonuclease

2.4 Primer extension

2.5 5′-nuclease

2.6 Oligonucleotide Ligation Assay

3 Other post-amplification methods based on physical properties of DNA

3.1 Single strand conformation polymorphism

3.2 Temperature gradient gel electrophoresis

3.3 Denaturing high performance liquid chromatography

3.4 High-resolution melting of the entire amplicon

3.5 Use of DNA mismatch-binding proteins

3.6 SNPlex (SNPlex™ is a proprietary genotyping platform sold by AppliedBiosystems).

Next-generation sequencing technologies such as pyrosequencing is alsouseful.

Reference is also made to GB2444410A and the genotyping method disclosedtherein, which is incorporated herein by reference in its entirety.

Miniaturized assays, such as microarrays with oligonucleotide reagentsimmobilized on small surfaces, are frequently proposed for large-scalemutation analysis and high-throughput genotyping (Large-scaleidentification, mapping, and genotyping of single-nucleotidepolymorphisms in the human genome (Wang D G, Fan J B, Siao C J, Berno A,Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J,Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E,Mittmann M, Morris M S, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S,Hudson T J, Lipshutz R, Chee M, Lander E S, Science. 1998 May 15;280(5366):1077-82). Other high-throughput methods discriminate allelesby differential hybridization, primer extension, ligation and cleavageof an allele-specific probe (Review Accessing genetic variation:genotyping single nucleotide polymorphisms, Syvanen A C, Nat Rev Genet.2001 December; 2(12):930-42; Review Techniques patents for SNPgenotyping, Twyman R M, Primrose S B, Pharmacogenomics. 2003 January;4(1):67-79).

An approach for a fully automated, large-scale SNP analysis is the‘homogeneous’ assay, i.e. a single-phase assay without separation steps,permitting continual monitoring during amplification. The TaqMan assay(Applied Biosystems), originally designed for quantitative real-timePCR, is a homogeneous, single-step assay also used in determination ofmutation status of DNA (see, eg, A. A. Komar (ed.), Single NucleotidePolymorphisms, Methods in Molecular Biology 578, DOI10.1007/978-1-60327-411-1_19, Humana Press, a part of SpringerScience+Business Media, LLC; and Single Nucleotide Polymorphisms,Methods in Molecular Biology™ Volume 578, 2009, pp 293-306, The TaqManMethod for SNP Genotyping, Gong-Qing Shen et al). The TaqMan SNPGenotyping Assay exploits the 5′-exonuclease activity of AmpliTaq GoldDNA polymerase to cleave a doubly labeled probe hybridized to theSNP-containing sequence of ssDNA. Cleavage separates a 5′-fluorophorefrom a 3′-quencher leading to detectable fluorescent signal. The use oftwo allele-specific probes carrying different fluorophores permits SNPdetermination in the same tube without any post-PCR processing. Genotypeis determined from the ratio of intensities of the two fluorescentprobes at the end of amplification. Thus, rather than taking advantageof the full set of real-time PCR data as in quantitative studies, onlyend-point data are used.

TaqMan SNP genotyping in a high-throughput, automated manner isfacilitated by the use of validated Pre-made TagMan© Genotyping assays,but Custom TagMan© Assays may also be used (High-throughput genotypingwith single nucleotide polymorphisms, Ranade K, Chang M S, Ting C T, PeiD, Hsiao C F, Olivier M, Pesich R, Hebert J, Chen Y D, Dzau V J, Curb D,Olshen R, Risch N, Cox DR, Botstein D, Genome Res. 2001 July;11(7):1262-8; Assessment of two flexible and compatible SNP genotypingplatforms: TaqMan SNP Genotyping Assays and the SNPlex GenotypingSystem, De la Vega F M, Lazaruk K D, Rhodes M D, Wenz M E I, Mutat Res.2005 Jun. 3; 573(1-2):111-35). The results of the assay can beautomatically determined by genotyping software provided with real-timethermal cyclers (e.g. IQ software of Bio-Rad, Sequence DetectionSoftware of Applied Biosystems).

Single nucleotide polymorphisms (SNPs) can be determined using TaqManreal-time PCR assays (Applied Biosystems) and commercial software thatassigns genotypes based on reporter probe signals at the end ofamplification. An algorithm for automatic genotype caling of SNPs usingthe full course of TaqMan real-time data is available for use (A.Callegaro et al, Nucleic Acids Res. 2006; 34(7): e56, Published online2006 Apr. 14. doi: 10.1093/nar/gkl185, PMCID: PMC1440877). The algorithmis unique in that it classifies samples according to the behavior ofblanks (no DNA samples), which cluster with heterozygous samples. Thismethod of classification eliminates the need for positive controls andpermits accurate genotyping even in the absence of a genotype class, forexample when one allele is rare.

The skilled person will be familiar with techniques that can be used foraccurate phenotyping and application to the invention. These include theuse of amino acid sequencing of isolated target protein and comparisonof sequences from different variants (eg, with the most common variant).An antibody that specifically and selectively binds in the area of a SNPunder stringent conditions can also be used to identify a particularvariant. In another method, the genotype is determined and acorresponding amino acid sequence (phenotype) determined, eg, by insilico translation.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the anti-TOIligand, eg, antibodies or antigen-binding fragments thereof, of thepresent invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the ligand, eg, antibody, of the present invention isused for treating various conditions and diseases associated with theTOI in an adult patient, it is advantageous to intravenously administerthe antibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer theligand or pharmaceutical composition of the invention, for example aligand provided by e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the mutantviruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J.Biol. Chem. 262:4429-4432). Methods of introduction include, but are notlimited to, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The ligand orcomposition may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The ligand or pharmaceutical composition can be also delivered in avesicle, in particular a liposome (see Langer (1990) Science249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss,New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.).

In certain situations, the ligand or pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng.14:201). In another embodiment, polymeric materials can be used; see,Medical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974). In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138, 1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a ligand or pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the ligand(s). Such dosage forms in a unit doseinclude, for example, tablets, pills, capsules, injections (ampoules),suppositories, etc. The amount of the aforesaid antibody contained isgenerally about 5 to about 500 mg per dosage form in a unit dose;especially in the form of injection, it is preferred that the aforesaidantibody is contained in about 5 to about 100 mg and in about 10 toabout 250 mg for the other dosage forms.

Exemplary TOIs

For example in any configuration, aspect, concept, example orconfiguration of the invention, the or each TOI is selected from thegroup consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1;ADORA2A; Aggrecan; AGR2; AICDA; AW1; AIG1; AKAP1; AKAP2; MYTH;amyloid-beta; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC;APOC1; AR; Ax1; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF;BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); B1yS; BMP1; BMP2;BMP3B (GDF1O); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin);BRCA1; C19orflO (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1;CB1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4);CCL15 (MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19(MIP-3b); CCL2 (MCP-1); MCAF; CCL2O (MIP-3a); CCL21 (MIP-2); SLC;exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2 I eotaxin-2);CCL25 (TECK); CCL26 (eotaxin-3); CCL2? (CTACK/ILC); CCL28; CCL3(MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2);CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKR1/HM145); CCR2(mcp-1RB/RA);CCR3 (CKR3/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD7,CD164; CD19; CD1C; CD2O; CD200; CD-22; CD24; CD28; CD3; CD37; CD38;CD3E; CD3G; CD3Z; CD4; CD4O; CD40L; CD44; CD45RB; CD52; CD69; CD72;CD74; CD79A; CD79B; CD8; CD8O; CD81; CD83; CD86; CD96; CD207; CDH1(E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH2O; CDH5; CDH7;CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKNIA(p21Wap1/Cip1); CDKNIB (p27Kip1); CDKNIC; CDKN2A (p161NK4a); CDKN2B;CDKN2C; CDKN3; CEBPB; CELSR3; CER1; CHGA; CHGB; Chitinase; CHRNG;CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8;CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLR1; CMKOR1 (RDC1);CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CRLF2;CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsinB); CX3CL1 (SCYDi); CX3CR1 (V28); CXCR6; CXCL1 (GRO1); CXCL1O (IP-10);CXCL11 (I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2(GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG);CXCR3 (GPR9/CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1;CYSLTR1; DAB2IP; DAND5; DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1;EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3;EphA4; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD;FasL; FASN; FCER1A; FCER2; FCGR3A; FCRL4; FGF; FGF1 (aFGF); FGF1O;FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2(bFGF); FGF2O; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6(HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (EPSILON);FIL1 (ZETA); FLJ12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1(FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; Galectin-3;GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GHR; GM-CSF; GNAS1; GNRH1; GPR2(CCR1O); GPR31; GPR44; GPR81 (FKSG8O); GPR87; GPR137C; GRCC10 (C10);GRP; GSN (Gelsolin); GSTP1; HAVCR1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9;hepcidin; hemojuvelin; HGF; HIF1A; HIP1; histamine and histaminereceptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A; ICEBERG; ICOS; 1D2;IFN-α; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; TFNW1;IGBP1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA;IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13;IL13RA1; IL13RA2; IL14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R;1L18; IL18BP; IL18R1; IL18RAP; 1L19; IL1A; IL1B; IL1F1O; IL1F5; IL1F6;IL1F7; IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2;IL1RL1;IL1RL2 IL1RN; 1L2; 1L20; IL2ORA; IL21R; 1L22; 1L22R; 1L22RA2;1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L28B; 1L29; IL2RA; IL2RB; IL2RG;1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6 receptor; IL6ST(glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK;INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1; JAK1; JAK3; JUN;K6HF; KAI1; KDR; MTLG; KLF5 (GC Box BP); KLF6; KLK10; KLK12; KLK13;KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19);KRT2A; KRTHB6 (hair-specific type II keratin); LAG3; LAMAS; LEP(leptin); LIGHT; Lingo-p75; Lingo-Troy; LPS; LRP5; LTA (TNF-b); LTB;LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun);MDK; MIB1; midkine; MIF; MIP-2; MK167 (Ki-67); MMP2; MMP9; MS4A1; MSMB;MT3 (metallothionectin-ifi); MTSS 1; MUC 1 (mucin); MYC; MYD88; NCK2;neurocan; Na_(v)1.7; Na_(v)1.8; NFKB1; NFKB2; NGFB (NGF); NGFR;NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5;NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3;NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1;NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPG;OPRD1; OX4OL; OX40; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PCSK9,PD-1, PD-L1; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan;PIAS2; Placental Growth Factor (PIGF); PIK3CG; PLAU (uPA); PLG; PLXDC1;PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA;PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13;RGS3; RNF110 (ZNF144); ROBO2; ROR1; S100A2; SCGB1D2 (lipophilin B);SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelialMonocyte-activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5(maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1;SLC43A1; SLIT2; SPP1; SPRR1B (Spri); ST6GAL1; STAB1; STAT6; STEAP;STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2;TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1);THBS2; THBS4; THPO; TIE (Tie-i); TIM3; TMP3; tissue factor; TLR1O; TLR2;TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TMPRSS6; TNF; TNF-α; TNFAIP2(B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5;TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF1O (TRAIL); TNFSF11(TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L);TNFSF1 5 (VEGI); TNFSF18; TNFSF4 (0X40 ligand); TNFSF5 (CD4O ligand);TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD3O ligand); TNFSF9 (4-1BBligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase lia); TP53;TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TRAIL;TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGFA; VEGFB; VEGFC; versican; VHL C5;VLA-4; Wnt7A; XCL1 (lymphotactin); XCL2 (SCM-lb); XCR1 (GPR5/CCXCR1);YY1; and ZFPM2.

In an example, the TOI is a human TOI selected from Table 5.

In an example, the TOI is OX40 ligand.

In an example, the TOI is OX40.

In an example, the TOI is PCSK9.

In an example, the TOI is IL6 receptor (IL-6R).

In an example, the TOI is LIGHT.

In an example, the TOI is VEGF-A.

In an example, the TOI is TNF alpha.

In an example, the TOI is PIGF.

In an example, the TOI is IGF1R.

In an example, the TOI is OPG.

In an example, the TOI is ICOS

In an example, the TOI is NGF.

In an example, the TOI is BMP6.

In an example, the TOI is ferroportin.

In an example, the TOI is TMPRSS6.

In an example, the TOI is hemojuvelin.

In an example, the TOI is VEGF receptor.

In an example, the TOI is PDGF receptor.

In an example, the TOI is stem cell factor receptor.

In an example, the TOI is hepcidin.

In an example, the TOI is IL-4 receptor alpha.

In an example, the TOI is sclerostin.

In an example, the TOI is IL-13 receptor.

In an example, the TOI is CD7.

In an example, the TOI is delta-like ligand-4 (D114).

In an example, the TOI is HGF.

In an example, the TOI is angiopoietin-2 (Ang2).

In an example, the TOI is GDF8.

In an example, the TOI is ERBB3.

In an example, the TOI is IL-17 receptor.

In an example, the TOI is CD40.

In an example, the TOI is CD40 ligand.

In an example, the TOI is EGFR.

In an example, the TOI comprises a mutation as described herein for thatTOI or is encoded by a nucleotide sequence comprising a SNP as describedherein for that TOI. Optionally, additionally the human comprises saidTOI comprising said mutation; or the human comprises a nucleotidesequence encoding said TOI comprising said mutation.

For example, the TOI is human PCSK9 and comprises a mutation I474,E670G, N425S or Q619P in SEQ ID NO: 1. Optionally, additionally thehuman comprises said PCSK9 comprising said mutation; or the humancomprises a nucleotide sequence encoding said PCSK9 comprising saidmutation. In an alternative, for example herein where a PCSK9 is statedto be of a specific “form”, eg, form f, c, r, p, e, h, aj or q, in thealternative the PCSK9 is a human PCSK9 comprising a mutation R46L, A53V,N425S, A443T, I474V, Q619P and E670G (eg, comprises a mutation I474,E670G, N425S or Q619P) in SEQ ID NO: 1; for example, the PCSK9 comprisesI474V in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 ora nucleotide sequence encoding such a PCSK9 (eg, wherein the method isfor treating or preventing dislipidemia, eg reducing cholesterol ormaintaining reduced cholesterol in the human); for example, the PCSK9comprises E670G in SEQ ID NO: 1 and optionally the human comprises sucha PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein themethod is for treating or preventing dislipidemia, eg reducingcholesterol or maintaining reduced cholesterol in the human); forexample, the PCSK9 comprises Q619P in SEQ ID NO: 1 and optionally thehuman comprises such a PCSK9 or a nucleotide sequence encoding such aPCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg reducing cholesterol or maintaining reduced cholesterolin the human); for example, the PCSK9 comprises N425S in SEQ ID NO: 1and optionally the human comprises such a PCSK9 or a nucleotide sequenceencoding such a PCSK9 (eg, wherein the method is for treating orpreventing dislipidemia, eg reducing cholesterol or maintaining reducedcholesterol in the human); for example, the PCSK9 comprises R46L in SEQID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotidesequence encoding such a PCSK9 (eg, wherein the method is for treatingor preventing dislipidemia, eg increaing cholesterol in the human); forexample, the PCSK9 comprises A53V in SEQ ID NO: 1 and optionally thehuman comprises such a PCSK9 or a nucleotide sequence encoding such aPCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg increaing cholesterol in the human); for example, thePCSK9 comprises A443T in SEQ ID NO: 1 and optionally the human comprisessuch a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, whereinthe method is for treating or preventing dislipidemia, eg increaingcholesterol in the human).

For example, the TOI is human IL6R and comprises a mutation D358A orV385I in SEQ ID NO: 78. Optionally, additionally the human comprisessaid IL6R comprising said mutation; or the human comprises a nucleotidesequence encoding said IL6R comprising said mutation.

For example, the TOI is human IL4Ra and comprises a mutation selectedfrom the group consisting of I75V, E400A, C431R, S503P, Q576R and S752Ain SEQ ID NO: 67. Optionally, additionally the human comprises saidIL4Ra comprising said mutation; or the human comprises a nucleotidesequence encoding said IL4Ra comprising said mutation.

For example, the TOI is huma Nav1.7 and comprises a mutation selectedfrom the group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693 X (wherein X is an amino acid other than E), 767X (wherein X isan amino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G, eg, the mutation is 1161W.Optionally, additionally the human comprises said Nav1.7 comprising saidmutation; or the human comprises a nucleotide sequence encoding saidNav1.7 comprising said mutation.

For example, the TOI is human VEGF-A and is encoded by a VEGF-Anucleotide sequence comprising a SNP selected from the group consistingof rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323,rs1413711, rs833068, rs833069, rs3025000 and rs1570360. Optionally,additionally the human comprises said VEGF-A comprising said mutation;or the human comprises a nucleotide sequence encoding said VEGF-Acomprising said mutation.

For example, the TOI is human PDGF-B and is encoded by a PDGF-Bnucleotide sequence comprising a SNP selected from the group consistingof rs142404523 (ie, a C corresponding to position −776) and a Cat theposition of rs1800818 (ie, a C corresponding to position −735).Optionally, additionally the human comprises said PDGF-B comprising saidmutation; or the human comprises a nucleotide sequence encoding saidPDGF-B comprising said mutation.

For example, the TOI is human PDGFR-B and is encoded by a PDGFR-Bnucleotide sequence comprising a SNP selected from the group consistingof rs246395 (ie, a G corresponding to position 2601) and rs74943037 (ie,a T corresponding to position 1391). Optionally, additionally the humancomprises said PDGFR-B comprising said mutation; or the human comprisesa nucleotide sequence encoding said PDGFR-B comprising said mutation.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, or “reduction” are all used herein tomean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” typically means adecrease by at least 10% as compared to a reference level (e.g. theabsence of a given treatment) and can include, for example, a decreaseby at least about 10%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or more. As used herein, “reduction” doesnot encompass a complete reduction as compared to a reference level. Adecrease can be preferably down to a level accepted as within the rangeof normal for an individual without a given disorder. However, forexample, for the purposes of lowering or reducing cholesterol level, forexample, a reduction by about 5-10 points can be considered a “decrease”or “reduction.”

In certain aspects of all embodiments of the invention, the term“inhibition” is used. Inhibition refers and refers to decrease by atleast 10% as compared to a reference level (e.g. the absence of a giventreatment) and can include, for example, a decrease by at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or more including 100% inhibition as compared to areference level. “Complete inhibition” refers to a 100% inhibition ascompared to a reference level.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, an “increase” is a statistically significant increasein such level.

As used herein, the term “substantially” refers to the qualitativecondition of exhibiting total or near-total extent or degree of acharacteristic or property of interest. One of ordinary skill in thebiological arts will understand that biological and chemical phenomenararely, if ever, go to completion and/or proceed to completeness orachieve or avoid an absolute result. The term “substantially” istherefore used herein to capture the potential lack of completenessinherent in many biological and chemical phenomena. For the removal ofdoubt, “substantially” can refer to at least a 90% extent or degree of acharacteristic or property of interest, e.g. at least 90%, at least 92%,at least 95%, at least 98%, at least 99% or greater.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. In some embodiments, thesubject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,” “patient” and “subject” are used interchangeably herein.In some embodiments, the subject can be a non-human vertebrate, e.g. aprimate, a rodent, a mouse, a rat, a pig, a sheep, a zebrafish, a frog,etc.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of adisease or condition, e.g., a cardiovascular condition. A subject can bemale or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatmentor one or more complications related to such a condition, andoptionally, have already undergone treatment for the condition or theone or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” or “human in need” of treatment for a particularcondition can be a subject having that condition, such as increasedcholesterol levels, diagnosed as having that condition, or at risk ofdeveloping that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids with natural aminoacids. When referring to “modified polypeptides” one refers topolypeptides that include modified amino acids (e.g., phosphorylated,glycated, glycosylated, etc.) and amino acid analogs, regardless of itssize or function. “Protein” and “polypeptide” are often used inreference to relatively large polypeptides, whereas the term “peptide”is often used in reference to small polypeptides, but usage of theseterms in the art overlaps. The terms “protein” and “polypeptide” areused interchangeably herein when referring to a gene product andfragments thereof. Thus, exemplary polypeptides or proteins include geneproducts, naturally occurring proteins with the specified sequence. Onecan also use peptide homologs, peptide orthologs, peptide paralogs,peptide fragments and other equivalents, variants, fragments, andanalogs of the peptides as these terms are understood by one of ordinaryskill in the art.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one nucleic acid strand of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the nucleic acid can be DNA. Inanother aspect, the nucleic acid can be RNA. Suitable nucleic acidmolecules are DNA, including genomic DNA or cDNA. Other suitable nucleicacid molecules are RNA, including mRNA. In some aspects one can also useanalogs of nucleic acids.

As used herein, the term “nucleic acid probe” refers to an isolatedoligonucleotide molecule having a nucleic acid sequence which canhybridize to a target nucleic acid sequence, e.g. specifically hybridizeto the target sequence. In some embodiments, a nucleic acid probe canfurther comprise a detectable label. In some embodiments, a nucleic acidprobe can be attached to a solid surface. In some embodiments, a nucleicacid from is from about 5 nt to about 100 nt in length.

As used herein, the term “siRNA” refers to a nucleic acid that forms anRNA molecule comprising two individual strands of RNA which aresubstantially complementary to each other. Typically, the siRNA is atleast about 15-40 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is about 15-40 nucleotides inlength, and the double stranded siRNA is about 15-40 base pairs inlength, preferably about 19-25 base nucleotides, e.g., 19, 20, 21, 22,23, 24, or 25 nucleotides in length). In some embodiments, a siRNA canbe blunt-ended. In some embodiments, a siRNA can comprise a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. The siRNA moleculescan also comprise a 3′ hydroxyl group. In some embodiments, the siRNAcan comprise a 5′ phosphate group. A siRNA has the ability to reduce orinhibit expression of a gene or target RNA when the siRNA is present orexpressed in the same cell as the target gene, e.g. the target RNA.siRNA-dependent post-transcriptional silencing of gene expressioninvolves cutting the target RNA molecule at a site guided by the siRNA.

As used herein, “PCSK9” or “proprotein convertase subtilisin/kexin type9” refers to a serine protease involved in regulating the levels of thelow density lipoprotein receptor (LDLR) protein (Horton et al., 2007;Seidah and Prat, 2007). PCSK9 has been shown to directly interact withthe LDLR protein, be endocytosed along with the LDLR, andco-immunofluoresce with the LDLR throughout the endosomal pathway(Lagace et al., 2006). PCSK9 is a prohormone-proprotein convertase inthe subtilisin (S8) family of serine proteases (Seidah et al., 2003).The sequence of PCSK9 for a variety of species is known, e.g., humanPCSK9 (NCBI Gene ID No: 255738). Nucleotide and polypeptide sequencesfor a number of PCSK9 isoforms are provided herein, e.g., SEQ ID NOs:1-37.

PCSK9 exists as both a pro-form and a mature form. Autocatalysis of thePCSK9 proform occurs between Gln152 and Ser153 (VFAQ|SIP (SEQ ID NO:116)) (Naureckiene et al., 2003), and has been shown to be required forits secretion from cells (Seidah et al., 2003). The inactive form priorto this cleavage can be referred to herein as the “inactive”,“pro-form”, or “unprocessed” form of PCSK9. The C-terminal fragmentgenerated by the autocatalysis event can be referred to herein as the“mature,” “cleaved”, “processed” or “active” PCSK9. Examples of pro-formand mature PCSK9 isoforms are provided herein, see, e.g. SEQ ID NOs:1-27.

As used herein, the “catalytic domain” of PCSK9 refers to the portion ofa PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9, e.g.of SEQ ID NO: 1. As used herein, the “C-terminal domain” of PCSK9 refersto the portion of a PCSK9 polypeptide corresponding to positions 450-692of PCSK9, e.g., of SEQ ID NO: 1.

As used herein, a disease or condition “mediated by PCSK9” refers to adisease or condition which is caused by or characterized by a change inPCSK9, e.g. a change in expression level, a change in activity, and/orthe presence of a variant or mutation of PCSK9. Non-limiting examples ofsuch diseases or conditions can include, for example, a lipid disorder,hyperlipoproteinemia, hyperlipidemia; dyslipidemia;hypercholesterolemia, a heart attack, a stroke, coronary heart disease,atherosclerosis, peripheral vascular disease, claudication (eg,claudication associated with elevated cholesterol), type II diabetes,high blood pressure, and a cardiovascular disease or condition. Anotherexample is acute coronary syndrome. In an example, the disease orcondition is an inflammatory or autoimmune disease or condition. Methodsof identifying and/or diagnosing such diseases and conditions are wellknown to medical practitioners of ordinary skill.

A subject at risk of having or developing a disease or conditionmediated by PCSK9 can be a subject exhibiting one or more signs orsymptoms of such a disease or condition or having one or more riskfactors for such a disease or condition, e.g. being overweight, havingelevated cholesterol level, comprising one or more genetic polymorphismsknown to predispose to the disease or condition, e.g., elevatedcholesterol level, such as having a mutation in the LDLR (encodinglow-density lipoprotein receptor) or APOB (encoding apolipoprotein B) orin the PCSK9 gene and/or having a family history of such a disease orcondition.

As used herein, “ligand” refers to a molecule which can bind, e.g.,specifically bind, to a second molecule or receptor. In someembodiments, a ligand can be, e.g., an antibody, antibody fragment,antibody portion, and/or affibody.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the polypeptide or nucleic acid (eg, the most commonone in humans, eg, most frequent in a database as disclosed herein, suchas the 1000 Genomes Project database) by one or more amino acid ornucleic acid deletions, additions, yet retains one or more specificfunctions or biological activities of the naturally occurring molecule.Amino acid substitutions include alterations in which an amino acid isreplaced with a different naturally-occurring amino acid residue. Suchsubstitutions may be classified as “conservative”, in which case anamino acid residue contained in a polypeptide is replaced with anothernaturally occurring amino acid of similar character either in relationto polarity, side chain functionality or size. Such conservativesubstitutions are well known in the art. Substitutions encompassed bythe present invention may also be “non-conservative”, in which an aminoacid residue which is present in a peptide is substituted with an aminoacid having different properties, such as naturally-occurring amino acidfrom a different group (e.g., substituting a charged or hydrophobicamino; acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. In some embodiments amino acid substitutions areconservative. Also encompassed within the term variant when used withreference to a polynucleotide or polypeptide, refers to a polynucleotideor polypeptide that can vary in primary, secondary, or tertiarystructure, as compared to a reference polynucleotide or polypeptide,respectively (e.g., as compared to a wild-type polynucleotide orpolypeptide).

Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 caninclude the forms described herein as a, f, c, r, p, m, e h, aj, and q.Sequences of these variants are provided herein, see, e.g, SEQ IDNOs:1-27 and in Table 1, 2 or 6.

In some aspects, one can use “synthetic variants”, “recombinantvariants”, or “chemically modified” polynucleotide variants orpolypeptide variants isolated or generated using methods well known inthe art. “Modified variants” can include conservative ornon-conservative amino acid changes, as described below. Polynucleotidechanges can result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence. Some aspects use include insertion variants, deletion variantsor substituted variants with substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. The term “conservativesubstitution,” when describing a polypeptide, refers to a change in theamino acid composition of the polypeptide that does not substantiallyalter the polypeptide's activity. For example, a conservativesubstitution refers to substituting an amino acid residue for adifferent amino acid residue that has similar chemical properties.Conservative amino acid substitutions include replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, or athreonine with a serine.

“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the blood brainbarrier (BBB)). Conservative substitution tables providing functionallysimilar amino acids are well known in the art. For example, thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company(1984), incorporated by reference in its entirety.) In some embodiments,individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids can alsobe considered “conservative substitutions” if the change does not reducethe activity of the peptide. Insertions or deletions are typically inthe range of about 1 to 5 amino acids. The choice of conservative aminoacids may be selected based on the location of the amino acid to besubstituted in the peptide, for example if the amino acid is on theexterior of the peptide and expose to solvents, or on the interior andnot exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e. its exposure to solvents (i.e. if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art, for example as disclosed in Dordo et al, J.MoI Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol.119(1986);205-218 and S. French and B. Robson, J. MoI. Evol.19(1983)171. Accordingly, one can select conservative amino acidsubstitutions suitable for amino acids on the exterior of a protein orpeptide (i.e. amino acids exposed to a solvent), for example, but notlimited to, the following substitutions can be used: substitution of Ywith F, T with S or K, P with A, E with D or Q, N with D or G, R with K,G with N or A, T with S or K, D with N or E, I with L or V, F with Y, Swith T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acidsubstitutions encompassed suitable for amino acids on the interior of aprotein or peptide, for example one can use suitable conservativesubstitutions for amino acids is on the interior of a protein or peptide(i.e. the amino acids are not exposed to a solvent), for example but notlimited to, one can use the following conservative substitutions: whereY is substituted with F, T with A or S, I with L or V, W with Y, M withL, N with D, G with A, T with A or S, D with N, I with L or V, F with Yor L, S with A or T and A with S, G, T or V. In some embodiments,non-conservative amino acid substitutions are also encompassed withinthe term of variants.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulphide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria. Antibodies can be humanized usingroutine technology.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody fragment” refers to a polypeptidethat includes at least one immunoglobulin variable domain orimmunoglobulin variable domain sequence and which specifically binds agiven antigen. An antibody fragment can comprise an antibody or apolypeptide comprising an antigen-binding domain of an antibody. In someembodiments, an antibody fragment can comprise a monoclonal antibody ora polypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody fragment” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesand combinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). VH refers to the variable domainof the heavy chain. VL refers to the variable domain of the light chain.According to the methods used in this invention, the amino acidpositions assigned to CDRs and FRs may be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)) or according to IMGTnomenclature.

D domain or region refers to the diversity domain or region of anantibody chain. J domain or region refers to the joining domain orregion of an antibody chain

An antibody “gene segment”, e.g. a VH gene segment, D gene segment, orJH gene segment refers to oligonucleotide having a nucleic acid sequencethat encodes that portion of an antibody, e.g. a VH gene segment is anoligonucleotide comprising a nucleic acid sequence that encodes apolypeptide VH domain.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, IMGT or Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality.

As used herein, the term “antibody binding site” refers to a polypeptideor domain that comprises one or more CDRs of an antibody and is capableof binding an antigen. For example, the polypeptide comprises a CDR3(eg, HCDR3). For example the polypeptide comprises CDRs 1 and 2 (eg,HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (eg,HCDRs1-3). In an example, the antibody binding site is provided by asingle variable domain (eg, a VH or VL domain). In another example, thebinding site comprises a VH/VL pair or two or more of such pairs.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. For example, in an diagnostic test the specific binding of aligand can distinguish between two variant PCSK9 proteins as describedherein. In some embodiments, specific binding can refer to an affinityof the first entity for the second target entity which is at least 10times, at least 50 times, at least 100 times, at least 500 times, atleast 1000 times or greater than the affinity for the third nontargetentity. In the context of oligonucleotide strands which interact viahybridization, specific binding can be “specific hybridization.”

Additionally, and as described herein, a recombinant human(ized)antibody can be further optimized to decrease potential immunogenicity,while maintaining functional activity, for therapy in humans. In thisregard, functional activity means a polypeptide capable of displayingone or more known functional activities associated with a recombinantantibody or antibody reagent thereof as described herein. Suchfunctional activities include, e.g. the ability to bind to a targetmolecule.

The term “immunizing” refers to the step or steps of administering oneor more antigens to an animal so that antibodies can be raised in theanimal Generally, immunizing comprises injecting the antigen or antigensinto the animal. Immunization can involve one or more administrations ofthe antigen or antigens. Suitable methods are prime-boost and RIMMSprocedures as known to the skilled person in the art.

As used herein, an “affibody” refers to a relatively small syntheticprotein molecule that has high binding affinity for a target protein(e.g. for PCSK9 or a variant therefo). Affibodies are composed of athree-helix bundle domain derived from the IgG-binding domain ofstaphylococcal protein A. The protein domain consists of a 58 amino acidsequence, with 13 randomized amino acids affording a range of affibodyvariants. Despite being significantly smaller than an antibody (anaffibody weighs about 6 kDa while an antibody commonly weighs about 150kDa), an affibody molecule works like an antibody since its binding siteis approximately equivalent in surface area to the binding site of anantibody.

As used herein, “VH3-23*04” refers to a human VH domain variantcomprising the polypeptide sequence of SEQ ID NO: 38. As opposed to thereference sequence, VH3-23*04 has a valine residue instead of a leucineresidue (see FIGS. 3 and 4; L24V, numbering including signal sequence;valine at position 5 shown in FIG. 4) as a result of the presence of thers56069819 SNP in the nucleic acid sequence encoding the VH domain. Asused herein, “rs56069819” refers to a mutation or variant in a VH genesegment from adenosine to cytosine (or thymine to guanine, dependingupon the strand of DNA which is being read), resulting in the VH domainencoding VH3-23*04. Rs56069819 is depicted in FIG. 4 and SEQ ID NO: 39,which demonstrate the T→G mutation (it is noted that the dbSNP entry forRS5606819 depicts the other strand, which comprises the A→C mutation).Further description of VH3-23*04 can be found, e.g., in US PatentPublication 2013/0071405; which is incorporated by reference herein inits entirety.

As used herein, “determine” or “determining” refers to ascertaining,e.g., by a quantitative or qualitative analysis. As used herein, “hasbeen determined” can refer to ascertaining on the basis of previouslyobtained information or simultaneously obtained information.

In some aspects of all embodiments of the invention selecting caninclude automation such as a computer implemented software program thatupon input of the relevant data such as ethnicity or a panel of SNP datacan make the determination based on the instructions set forth herein.

As used herein, “assaying” refers to assessing, evaluating, quantifying,measuring, or characterizing an analyte, e.g., measuring the level of ananalyte in a sample, identifying an analyte, or detecting the presenceor absence of an analyte in a sample. In some embodiments, assayingrefers to detecting a presence or absence of the analyte of interest. Insome embodiments, assaying refers to quantifying an amount of ananalyte, e.g., providing a measure of concentration or degree of analyteabundance. In some embodiments, assaying refers to enumerating thenumber of molecules of analyte present in a sample and/or specimen,e.g., to determine an analyte copy number.

As used herein “multiplex” refers to the carrying out of a method orprocess simultaneously and in the same reaction vessel on two or more,typically three or more, different target sequences, e.g. on two or moreisoforms of PCSK9, or PCSK9 and an additional target. A multiplexanalysis typically includes analysis of 10-50; 10-100; 104000, 10-5000,10-10000 reactions in a multiplex format, such as a multiwall, an array,or a multichannel reaction.

Often the analysis or multiplex analysis is also automated usingrobotics and typically software executed by a computer and may include arobotic handling of samples, automatic or robotic selection of positiveor negative results, assaying for presence of absence of a target, suchas a nucleic acid polymorphism or a protein variant.

The term “biological sample” or “test sample” as used herein denotes asample taken or isolated from a biological organism, e.g., a sample froma subject. Exemplary biological samples include, but are not limited to,a biofluid sample; serum; plasma; urine; saliva; hair, epithelial cells,skin, a tumor biopsy and/or tissue sample etc. The term also includes amixture of the above-mentioned samples. The term “test sample” or“biological sample” also includes untreated or pretreated (orpre-processed) biological samples. For the analysis of nucleic acids,the biological sample should typically comprise at least one cellcomprising nucleic acids.

The test sample can be obtained by removing a sample of cells from asubject, but can also be accomplished by using previously isolated cells(e.g. isolated at a prior time point and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected, refrigerated, frozen or otherwise preservedsample.

In some embodiments, the test sample can be an untreated test sample. Asused herein, the phrase “untreated test sample” refers to a test samplethat has not had any prior sample pre-treatment except for dilutionand/or suspension in a solution. Exemplary methods for treating a testsample include, but are not limited to, centrifugation, filtration,sonication, homogenization, heating, freezing and thawing, andcombinations thereof. In some embodiments, the test sample can be afrozen test sample, e.g., a frozen tissue. The frozen sample can bethawed before employing methods, assays and systems described herein.After thawing, a frozen sample can be centrifuged before being subjectedto methods, assays and systems described herein. In some embodiments,the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments, a test sample can be a pre-processedtest sample, for example, supernatant or filtrate resulting from atreatment selected from the group consisting of centrifugation,filtration, thawing, purification, and any combinations thereof. In someembodiments, the test sample can be treated with a chemical and/orbiological reagent. Chemical and/or biological reagents can be employedto protect and/or maintain the stability of the sample, includingbiomolecules (e.g., nucleic acid and protein) therein, duringprocessing. One exemplary reagent is a protease inhibitor, which isgenerally used to protect or maintain the stability of protein duringprocessing. The skilled artisan is well aware of methods and processesappropriate for pre-processing of biological samples required fordetermination of the level of an expression product as described herein.

As used herein, “genotyping” refers to a process of determining thespecific allelic composition of a cell and/or subject at one or moreposition within the genome, e.g. by determining the nucleic acidsequence at that position. Genotyping refers to a nucleic acid analysisand/or analysis at the nucleic acid level. As used herein, “phenotyping”refers a process of determining the identity and/or composition of anexpression product of a cell and/or subject, e.g. by determining thepolypeptide sequence of an expression product. Phenotyping refers to aprotein analysis and/or analysis at the protein level.

As used herein, the term “nucleic acid amplification” refers to theproduction of additional copies of a nucleic acid sequence and istypically carried out using polymerase chain reaction (PCR) or ligasechain reaction (LCR) technologies well known in the art (Dieffenbach, C.W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview, N. Y.). Other methods for amplificationare also contemplated in aspects of the invention.

The term “allele-specific amplification” refers to a reaction (e.g., PCRreaction) in which at least one of the primers (e.g., allele-specificprimer) is chosen from a polymorphic area of gene (e.g., singlenucleotide polymorphism), with the polymorphism located at or near theprimer's 3′-end. A mismatched primer will not initiate amplification,whereas a matched primer will initiate amplification. The appearance ofan amplification product is indicative of the presence of thepolymorphism.

As used herein, “sequencing” refers to the determination of the exactorder of nucleotide bases in a strand of DNA (deoxyribonucleic acid) orRNA (ribonucleic acid) or the exact order of amino acids residues orpeptides in a protein. Nucleic acid sequencing can be done using Sangersequencing or next-generation high-throughput sequencing.

As used herein “next-generation sequencing” refers to oligonucleotidesequencing technologies that have the capacity to sequenceoligonucleotides at speeds above those possible with conventionalsequencing methods (e.g. Sanger sequencing), due to performing andreading out thousands to millions of sequencing reactions in parallel.Non-limiting examples of next-generation sequencing methods/platformsinclude Massively Parallel Signature Sequencing (Lynx Therapeutics); 454pyro-sequencing (454 Life Sciences/Roche Diagnostics); solid-phase,reversible dye-terminator sequencing (Solexa/Illumina): SOLiD technology(Applied Biosystems); Ion semiconductor sequencing (ION Torrent); DNAnanoball sequencing (Complete Genomics); and technologies available fromPacific Biosciences, Intelligen Bio-systems, Oxford NanoporeTechnologies, and Helicos Biosciences. Next-generation sequencingtechnologies and the constraints and design parameters of associatedsequencing primers are well known in the art (see, e.g. Shendure, etal., “Next-generation DNA sequencing,” Nature, 2008, vol. 26, No. 10,1135-1145; Mardis, “The impact of next-generation sequencing technologyon genetics,” Trends in Genetics, 2007, vol. 24, No. 3, pp. 133-141; Su,et al., “Next-generation sequencing and its applications in moleculardiagnostics” Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al.,“The impact of next-generation sequencing on genomics”, J GenetGenomics, 2011, 38(3):95-109; (Nyren, P. et al. Anal Biochem 208: 17175(1993); Bentley, D. R. Curr Opin Genet Dev 16:545-52 (2006); Strausberg,R. L., et al. Drug Disc Today 13:569-77 (2008); U.S. Pat. Nos.7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; 6,818,395;6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349;which are incorporated by reference herein in their entireties).

As used herein, “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands. In some embodiments, nucleic acidhybridization can refer to a method of determining a nucleic acidsequence and/or identity by hybridizing a nucleic acid sample with aprobe, e.g. Northern or Southern blot analysis or microarray analysis.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment). For treatment to beeffective a complete cure is not contemplated. The method can in certainaspects include cure as well.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

Multiple compositions can be administered separately or simultaneously.Separate administration refers to the two compositions beingadministered at different times, e.g. at least 10, 20, 30, or 10-60minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One canalso administer compositions at 24 hours apart, or even longer apart.Alternatively, two or more compositions can be administeredsimultaneously, e.g. less than 10 or less than 5 minutes apart.Compositions administered simultaneously can, in some aspects, beadministered as a mixture, with or without similar or different timerelease mechanism for each of the components.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one complex, enzyme, orcell. Exemplary delivery methods include, but are not limited to, directdelivery to cell culture medium, perfusion, injection, or other deliverymethod well known to one skilled in the art.

As used herein, “obtain” refers to any method of acquiring, securing,procuring, or coming into the possession of, e.g. a sample. Obtaining abiological sample from a subject can comprise physical removing a samplefrom a subject (e.g. drawing blood or taking a hair or saliva sample)without or without active participation from the subject; receiving asample from a subject (e.g. the subject collects a saliva or hair samplethemselves and provides it, e.g. in a container provided for thepurpose); or procuring a sample from a storage facility, medicalfacility, or medical provider. Obtain from the human or subject, refersto an active step of, e.g., drawing blood or taking a tissue or cellsample.

As used herein, “cholesterol level” refers to a level of one or more oftotal cholesterol, LDL cholesterol, HDL cholesterol, and/ortriglycerides. Cholesterol levels can be the level of cholesterol in theblood of a subject.

As used herein in reference to cholesterol levels, “maintain” refers topreventing the level from worsening (e.g. increasing). In someembodiments, maintaining a particular level refers to a process thatresults in the cholesterol level not increasing by more than 10% overtime. Maintaining may also refer to maintaining a previously achievedlevel. For example, if a human has received statin treatment, one canmaintain the cholesterol level achieved using the statin treatment.

In some embodiments, the subject treated according to the methodsdescribed herein has previously had their cholesterol level reduced. Asused herein, “previously reduced” indicates that at a prior point intime, the subject experienced a decrease in cholesterol levels. Thedecrease can be due to administration of a pharmaceutical composition(e.g. administration of a composition as described herein or anothercomposition, e.g. a statin) or due to another cause, e.g. a change indiet and/or exercise.

An existing treatment for high cholesterol levels is the administrationof a statin. As referred to herein, a “statin” (also known as HMG-CoAreductase inhibitors) are inhibitors of the enzyme HMG-coA reductase,which mediates cholesterol production in the liver. Statins, bycompetitively binding HMG-CoA reductase, prevent the binding of HMG-CoAto the enzyme and thereby inhibit the activity of the reductase (e.g.the production of mevalonate). Non-limiting examples of statins caninclude atorvastatin (LIPITOR™), fluvastatin (LESCOL™), lovastatin(MEVACOR™, ALTOCOR™), pitavastatin (LIVALO™), pravastatin (PRAVACHOL™)rosuvastatin (CRESTOR™), and simvastatin (ZOCOR™). Statins can beadministered in combination with other agents, e.g. the combination ofezetimibe and simvastatin.

Some subjects are, or become, resistant to statin treatment. As usedherein, “resistant to statin treatment” or “reduced responsiveness tostatin treatment” refers to a subject exhibiting a statisticallysignificantly lower response to the administration of a statin ascompared to a reference level. The reference level can be, e.g., theaverage response for a population of subjects or the level of theindividual subject at an earlier date. A response to statin treatment isreadily measured by one of skill in the art, e.g., measurement ofcholesterol levels, changes in cholesterol levels, and/or HMG-CoAreductase activity.

As used herein, the term “detectable label” refers to a molecule ormoiety that can be detected, e.g. measured and/or determined to bepresent or absent. Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments, detectable labels can include labels that can bedetected by spectroscopic, photochemical, biochemical, immunochemical,electromagnetic, radiochemical, or chemical means, such as fluorescence,chemifluoresence, or chemiluminescence, or any other appropriate means.The detectable labels used in the methods described herein can beprimary labels (where the label comprises a moiety that is directlydetectable or that produces a directly detectable moiety) or secondarylabels (where the detectable label binds to another moiety to produce adetectable signal, e.g., as is common in immunological labeling usingsecondary and tertiary antibodies). The detectable label can be linkedby covalent or non-covalent means to the reagent. Alternatively, adetectable label can be linked such as by directly labeling a moleculethat achieves binding to the reagent via a ligand-receptor binding pairarrangement or other such specific recognition molecules. Detectablelabels can include, but are not limited to radioisotopes, bioluminescentcompounds, chromophores, antibodies, chemiluminescent compounds,fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detectable label can be a fluorescentcompound. When the fluorescently label is exposed to light of the properwavelength, its presence can then be detected due to fluorescence. Insome embodiments, a detectable label can be a fluorescent dye molecule,or fluorophore including, but not limited to fluorescein, phycoerythrin,phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™,allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandemconjugates such as phycoerythrin-Cy5™, green fluorescent protein,rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™,rhodamine and derivatives (e.g., Texas red and tetrarhodimineisothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™,6-carboxyfhiorescein (commonly known by the abbreviations FAM and F),6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P. In some embodiments, a detectable label can be an enzyme including,but not limited to horseradish peroxidase and alkaline phosphatase. Anenzymatic label can produce, for example, a chemiluminescent signal, acolor signal, or a fluorescent signal. Enzymes contemplated for use as adetectable label can include, but are not limited to, malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,catalase, glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. In some embodiments, a detectable label is achemiluminescent label, including, but not limited to lucigenin,luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester. In some embodiments, a detectablelabel can be a spectral colorimetric label including, but not limited tocolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, and latex) beads.

In some embodiments, reagents can also be labeled with a detectable tag,such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detectionsystems can also be used, for example, a biotin-streptavidin system. Inthis system, the antibodies immunoreactive (i. e. specific for) with thebiomarker of interest is biotinylated. Quantity of biotinylated antibodybound to the biomarker is determined using a streptavidin-peroxidaseconjugate and a chromagenic substrate. Such streptavidin peroxidasedetection kits are commercially available, e. g. from DAKO; Carpinteria,Calif. A reagent can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the reagent using such metal chelating groupsas diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

As used herein, “authorization number” or “marketing authorizationnumber” refers to a number issued by a regulatory agency upon thatagency determining that a particular medical product and/or compositionmay be marketed and/or offered for sale in the area under the agency'sjurisdiction. As used herein “regulatory agency” refers to one of theagencies responsible for evaluating, e.g, the safety and efficacy of amedical product and/or composition and controlling the sales/marketingof such products and/or compositions in a given area. The Food and DrugAdministration (FDA) in the US and the European Medicines Agency (EPA)in Europe are but two examples of such regulatory agencies. Othernon-limiting examples can include SDA, MPA, MHPRA, IMA, ANMAT, Hong KongDepartment of Health-Drug Office, CDSCO, Medsafe, and KFDA.

As used herein, “injection device” refers to a device that is designedfor carrying out injections, an injection including the steps oftemporarily fluidically coupling the injection device to a person'stissue, typically the subcutaneous tissue. An injection further includesadministering an amount of liquid drug into the tissue and decoupling orremoving the injection device from the tissue. In some embodiments, aninjection device can be an intravenous device or IV device, which is atype of injection device used when the target tissue is the blood withinthe circulatory system, e.g., the blood in a vein. A common, butnon-limiting example injection device is a needle and syringe.

As used herein, a “buffer” refers to a chemical agent that is able toabsorb a certain quantity of acid or base without undergoing a strongvariation in pH.

As used herein, “packaging” refers to how the components are organizedand/or restrained into a unit fit for distribution and/or use. Packagingcan include, e.g., boxes, bags, syringes, ampoules, vials, tubes,clamshell packaging, barriers and/or containers to maintain sterility,labeling, etc.

As used herein, “instructions” refers to a display of written, printedor graphic matter on the immediate container of an article, for examplethe written material displayed on a vial containing a pharmaceuticallyactive agent, or details on the composition and use of a product ofinterest included in a kit containing a composition of interest,Instructions set forth the method of the treatment as contemplated to beadministered or performed.

As used herein, a “solid surface” refers to an object suitable for theattachment of biomolecules. Non-limiting examples of a solid surface caninclude a particle (including, but not limited to an agarose or latexbead or particle or a magnetic particle), a bead, a nanoparticle, apolymer, a substrate, a slide, a coverslip, a plate, a dish, a well, amembrane, and/or a grating. The solid surface can include many differentmaterials including, but not limited to, polymers, plastics, resins,polysaccharides, silicon or silica based materials, carbon, metals,inorganic glasses, and membranes.

As used herein, “classification” of a subject, e.g., classification ofthe subject's ancestry refers to determining if the subject hasbiological ancestors who originated in a particular geographical area,and are therefore likely to have particular genetic variants found inthe populations which have historically occupied that area.Classification can comprise, e.g. obtaining information on the subject'sfamily, interviewing the subject or a family member regarding theirbiological family's ancestry, and/or genetic testing. Classification canbe on the basis used for the 1000 Genomes Project, as will be familiarto the skilled person in the art. In some embodiments, the subject canbe classified as being of a particular ancestry if at least thesubject's genome comprises a substantial number of different alleles incommon with other humans of that ancestry (eg, determined by referenceto the 1000 Genomes Project database), for example, at least 10, 20, 30,40, 50 or 100 or more alleles in common. Abbreviations for particularancestral groups are provided in Table 4.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); BenjaminLewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10:0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology:a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998) which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

It will be understood that particular configurations, aspects, examples,clauses and embodiments described herein are shown by way ofillustration and not as limitations of the invention. The principalfeatures of this invention can be employed in various embodimentswithout departing from the scope of the invention. Those skilled in theart will recognize, or be able to ascertain using no more than routinestudy, numerous equivalents to the specific procedures described herein.Such equivalents are considered to be within the scope of this inventionand are covered by the claims. All publications and patent applicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference. The use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.” The use ofthe term “or” in the claims is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive, although the disclosure supports a definition that refers toonly alternatives and “and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variationof error for the device, the method being employed to determine thevalue, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

References to sequences by “SEQ ID NO:” herein (eg, in the claims) is byreference to the sequences appearing in the Tables; similarly referenceto position numbers “in SEQ ID NO:” (eg, in the claims) is by referenceto the sequences appearing in the Tables herein (eg, as per Tables 6, 10and 17). Thus, for example, “an Asp corresponding to position 204 of SEQID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42”refers to the Asp and Leu labelled as being at positions 204 and 206respectively of SEQ ID NO: 42 shown in Table 10 herein.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. selecting a human that is positive for the TOI polymorphic        variant, wherein the TOI in said human is encoded by a        nucleotide sequence comprising an allele having a cumulative        human allele frequency of less than 50% and/or wherein the TOI        in said human is encoded by a nucleotide sequence comprising an        allele having total human genotype frequency of less than 50%;        and    -   b. administering to the human an anti-TOI ligand to target the        TOI in the human to treat or prevent said disease or condition.        In a first alternative, paragraph 1 provides:—        A method of treating or preventing a disease or condition in a        human, wherein the disease or condition is mediated by a Target        of Interest (TOI), wherein the TOI is present in humans as        different polymorphic variants, the method comprising        administering to said human an anti-TOI ligand (eg, an antibody        or antibody fragment) that specifically binds a TOI variant that        is encoded by a nucleotide sequence comprising a SNP having a        cumulative human allele frequency of less than 50% (eg, less        than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%) and/or        wherein the TOI variant is encoded by a nucleotide sequence        comprising a SNP having total human genotype frequency of less        than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2,        or 1%); wherein the human comprises a (eg, said) TOI-encoding        nucleotide sequence comprising said SNP; wherein        (i) the ligand (eg, antibody or fragment) comprises a VH domain        derived from the recombination of a human VH segment, a human D        gene segment and a human JH segment, the human VH segment        comprising a SNP, and wherein the human comprises a (eg, said)        VH segment comprising said SNP; or        (ii) the ligand (eg, antibody or fragment) comprises a Vλ domain        derived from the recombination of a human Vλ segment and a human        Jλ segment, the human Vλ segment comprising a SNP, and wherein        the human comprises a (eg, said) Vλ segment comprising said SNP;        or        (iii) the ligand (eg, antibody or fragment) comprises a Vκ        domain derived from the recombination of a human Vκ segment and        a human Jκ segment, the human Vκ segment comprising a SNP, and        wherein the human comprises a (eg, said) Vκ segment comprising        said SNP; or        (iv) the ligand (eg, antibody or fragment) comprises a heavy        chain constant region (eg, a gamma-1, 2, 3o 4 constant region)        encoded by a nucleotide sequence comprising a SNP, and wherein        the human comprises a (eg, said) heavy chain constant region        (eg, a gamma-1, 2, 3 o 4 constant region respectively)        comprising said SNP; or        (v) the ligand (eg, antibody or fragment) comprises a lambda        chain constant encoded by a nucleotide sequence comprising a        SNP, and wherein the human comprises a (eg, said) lambda chain        constant region comprising said SNP; or        (vi) the ligand (eg, antibody or fragment) comprises a kappa        chain constant encoded by a nucleotide sequence comprising a        SNP, and wherein the human comprises a (eg, said) kappa chain        constant region comprising said SNP.        In a second alternative, paragraph 1 provides:—        A method of treating or preventing a disease or condition in a        human, wherein the disease or condition is mediated by a Target        of Interest (TOI), wherein the TOI is present in humans as        different polymorphic variants, the method comprising comprising        administering to said human an anti-TOI ligand (eg, an antibody        or antibody fragment) that specifically binds a TOI variant that        comprises a single amino acid variation (ie, a single amino acid        at a position where there is difference between TOI variants in        humans) having a cumulative human allele frequency of less than        50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or        1%) and/or wherein the amino acid variant has a total human        genotype frequency of less than 50% (eg, less than 40, 35, 30,        35, 20, 15, 10, 5, 4, 3, 2, or 1%); wherein the human expresses        TOI proteins comprising said single amino acid variation;        wherein        (i) the ligand (eg, antibody or fragment) comprises a VH domain        comprising the same single amino acid variation, and wherein the        human expresses VH domains comprising said single amino acid        variation; or        (ii) the ligand (eg, antibody or fragment) comprises a Vλ domain        comprising a single amino acid variation, and wherein the human        expresses Vλ domains comprising the same single amino acid        variation; or        (iii) the ligand (eg, antibody or fragment) comprises a Vκ        domain comprising a single amino acid variation, and wherein the        human expresses Vκ domains comprising the same single amino acid        variation; or        (iv) the ligand (eg, antibody or fragment) comprises a heavy        chain constant region (eg, a gamma-1, 2, 3 o 4 constant region,        eg, wherein the constant region is an Fc, CH1, CH2 or CH3)        comprising a single amino acid variation, and wherein the human        expresses a heavy chain constant region (eg, a gamma-1, 2, 3 o 4        constant region, eg, wherein the constant region is an Fc, CH1,        CH2 or CH3 respectively) comprising the same single amino acid        variation; or        (v) the ligand (eg, antibody or fragment) comprises a lambda        chain constant region comprising a single amino acid variation,        and wherein the human expresses a lambda chain constant region        comprising the same single amino acid variation; or        (vi) the ligand (eg, antibody or fragment) comprises a kappa        chain constant region comprising a single amino acid variation,        and wherein the human expresses a kappa chain constant region        comprising the same single amino acid variation.        The term “comprises a single amino acid variation” includes the        possibility of there being further amino acid variations in the        TOI sequence.        In an example, frequencies herein are according to the 1000        Genomes database, eg, the Phase I database or as shown in        Ensembl (available on the world wide web at ensembl.org), eg, at        1^(st) Oct. 2013 or 1^(st) Oct. 2014.        In an example, each SNP encodes a non-synonymous amino acid        variation, ie, the SNP is not a silent mutation.        Optionally, the method comprises selecting said human comprising        the nucleotide sequence prior to administration.        In a third alternative where the TOI is human PCSK9, paragraph 1        provides:—        A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a VH        domain derived from the recombination of a human VH segment, a        human D gene segment and a human JH segment, the human VH        segment encoding a valine at the amino acid corresponding to        position 5 of SEQ ID NO: 40 and wherein said human comprises (i)        a VH gene segment encoding the framework 1 of SEQ ID NO: 40        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.        In a fourth alternative where the TOI is human PCSK9, paragraph        1 provides:—        A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a VL        domain derived from the recombination of a human VL segment and        a human JL segment, the human VL segment is a Vλ or Vκ disclosed        herein and wherein said human comprises (i) said VL gene segment        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.        In a fifth alternative where the TOI is human PCSK9, paragraph 1        provides:—        A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation as defined herein (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody comprises a C        domain encoded by a human CH, Cλ or Cκ gene segment disclosed        herein and wherein said human comprises (i) said C gene segment        and (ii) a nucleotide sequence encoding a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation in SEQ ID NO: 1.        The following paragraphs 2-8, 19 and 49 onwards relate to any of        the alternatives of paragraph 1.-   2. The method of paragraph 1, wherein before step (a) the ligand has    been or is determined as being capable of specifically binding to    said TOI variant.-   3. The method of paragraph 1 or 2, comprising determining that the    human is positive for the TOI polymorphic variant, optionally    wherein the step of determining comprises determining that the human    is positive for a nucleotide variant encoding said TOI variant.-   4. The method of paragraph 3, wherein the step of determining    comprises assaying a biological sample obtained from said human for    a nucleotide polymorphism encoding said TOI polymorphic variant.-   5. The method of paragraph 3 or 4, wherein the step of determining    comprises assaying a biological sample obtained from said human for    a protein corresponding to the TOI polymorphic variant.-   6. The method of any preceding paragraph, wherein said frequency is    less than 15%.-   7. The method of any preceding paragraph, wherein said frequency is    less than 10%.-   8. The method of any preceding paragraph, wherein the ligand is    capable of specifically binding to two or more different TOI    variants, each being encoded by a nucleotide sequence comprising an    allele having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50%.-   9. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. selecting a human that is negative for a variant nucleotide        sequence comprising an allele having a cumulative human allele        frequency of less than 50% and/or having a total human genotype        frequency of less than 50%; or that the human is negative for a        TOI variant encoded by a nucleotide sequence comprising the        allele having a cumulative human allele frequency of less than        50% and/or having a total human genotype frequency of less than        50%; and    -   b. administering to the human an anti-TOI ligand to target the        TOI variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is a variant encoded by        a nucleotide sequence comprising an allele having a cumulative        human allele frequency of more than 50% and/or having a total        human genotype frequency of more than 50%.-   10. The method of paragraph 9, comprising determining that the human    is positive for the TOI polymorphic variant, optionally wherein the    determining comprises that the human has been or is phenotyped as    positive for the most frequent TOI variant or genotyped for the    nucleotide sequence thereof-   11. The method of paragraph 10, wherein determining comprises    assaying for the nucleotide sequence to determine the presence of    said allele.-   12. The method of paragraph 11, wherein the assaying comprises    nucleic acid amplification.-   13. The method of paragraph 11 or 12, wherein the assaying comprises    hybridization, sequencing, or next generation sequencing.-   14. The method of any of paragraphs 11-13, further comprising the    step of obtaining a biological sample from the human.-   15. The method of any one of paragraphs 9-14, wherein the ligand has    been or is determined as being capable of specifically binding to    the most frequent TOI variant.-   16. The method of any one of paragraphs 9-15, wherein the ligand has    been or is determined as being substantially incapable of    neutralising or inhibiting said TOI variant.-   17. The method of any one of paragraphs 9-16, wherein the ligand is    capable of specifically binding to the most frequent TOI variant.-   18. The method of any one of paragraphs 9-17, wherein the ligand is    capable of specifically binding to two or more different TOI    variants, each being encoded by a nucleotide sequence comprising an    allele having a cumulative human allele frequency of more than 50%.-   19. The method of any preceding paragraph, wherein said TOI    polymorphic variant has been or is determined as being present in at    least two different human ethnic populations.-   20. The method of any preceding paragraph, wherein said cumulative    human allele frequency is the frequency in a database of    naturally-occurring sequences sampled from at least 15 different    human ethnic populations and comprising at least 1000 sequences.-   21. The method of any of the preceding paragraphs, wherein the    ligand is an antibody, antibody fragment or an affibody.-   22. The method of any of the preceding paragraphs, wherein the    ligand comprises a nucleotide sequence that specifically hybridises    to a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50% or an RNA transcript    thereof; and/or the ligand comprises a nucleotide sequence that    comprises at least 10 contiguous nucleotides of a nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50% or    is an antisense sequence thereof-   23. The method of any of the preceding paragraphs, wherein the    genome of said human comprises an allele having a cumulative human    allele frequency of less than 50% and the allele is found in at    least 2 different ethnic populations.-   24. A composition comprising a ligand capable of binding a target of    interest encoded by a nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and the    allele is found in at least 2 different ethnic populations and    optionally a pharmaceutically acceptable carrier and optionally a    label or instructions for use to treat and/or prevent said disease    or condition in a human; optionally wherein the label or    instructions comprise a marketing authorisation number issued by a    regulatory authority.-   25. A kit for treating or preventing a condition or disease mediated    by a target of interest as recited in any preceding paragraph, the    kit comprising a ligand capable of specifically binding a target of    interest encoded by a nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and the    allele is found in at least 2 different ethnic populations; and    optionally in combination with a label or instructions for use to    treat and/or prevent said disease or condition in a human;    optionally wherein the label or instructions comprise a marketing    authorisation number issued by a regulatory agency; optionally    wherein the kit comprises an injection pen or IV container that    comprises the ligand.-   26. The composition of paragraph 24 or the kit of paragraph 25,    wherein the regulatory agency is FDA or EMA.-   27. A method of producing an anti-human TOI antibody binding site,    the method comprising obtaining a plurality of anti-TOI antibody    binding sites, screening the antibody binding sites for binding to a    TOI comprising an amino acid sequence encoded by a nucleotide    sequence comprising an allele having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%, or to a peptide thereof that comprises an amino    acid variation from the corresponding sequence encoded by the    TOI-encoding nucleotide sequence comprising an allele having the    highest cumulative human allele frequency and/or the highest total    human genotype frequency, and isolating an antibody binding site    that binds in the screening step.-   28. A method of producing an anti-human TOI antibody, the method    comprising immunising a non-human vertebrate with a TOI comprising    an amino acid sequence encoded by a nucleotide sequence comprising    an allele having a cumulative human allele frequency of less than    50% and/or a total human genotype frequency of less than 50%, or to    a peptide thereof that comprises an amino acid variation from the    corresponding sequence encoded by the TOI-encoding nucleotide    sequence comprising an allele having the highest cumulative human    allele frequency and/or the highest total human genotype frequency,    and isolating an antibody that binds a TOI comprising an amino acid    sequence encoded by a TOI nucleotide sequence comprising an allele    having a cumulative human allele frequency of less than 50% and/or a    total human genotype frequency of less than 50%, and optionally    producing a TOI-binding fragment or derivative of the isolated    antibody.-   29. The method of paragraph 28, wherein the non-human vertebrate is    a mouse or a rat.-   30. The method of paragraph 29 or 30, comprising the step of    obtaining a nucleic acid encoding the antibody, fragment, derivative    or binding site and optionally inserting the nucleic acid in an    expression vector.-   31. A kit for TOI genotyping a human, wherein the kit comprises a    nucleic acid comprising a nucleotide sequence that specifically    hybridises to a TOI nucleotide sequence selected having a cumulative    human allele frequency of less than 50% and/or a total human    genotype frequency of less than 50% or an RNA transcript thereof;    and/or the nucleic acid comprises a nucleotide sequence that    comprises at least 10 contiguous nucleotides of a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50% or is an    antisense sequence thereof-   32. A kit for TOI genotyping or phenotyping a human, wherein the kit    comprises a ligand capable of binding a target of interest encoded    by a nucleotide sequence comprising an allele having a cumulative    human allele frequency of less than 50% or an antibody, fragment or    derivative produced by the method of any one of paragraphs 28 to 30.-   33. The kit of paragraph 32, wherein the allele is found in at least    2 different ethnic populations.-   34. Use of an anti-TOI ligand that specifically binds a human TOI    comprising an amino acid sequence encoded by a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, in the    manufacture of a medicament for treating and/or preventing a    TOI-mediated disease or condition in a human whose genome comprises    a TOI nucleotide sequence having a cumulative human allele frequency    of less than 50% and/or having a total human genotype frequency of    less than 50%.-   35. Use of an anti-TOI ligand that specifically binds a human TOI    comprising an amino acid sequence encoded by a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, in the    manufacture of a medicament for targeting said TOI in a human to    treat and/or prevent a disease or condition mediated by TOI.-   36. A method of targeting a Target of Interest (TOI) for treating    and/or preventing a TOI-mediated disease or condition in a human,    the method comprising administering an anti-TOI ligand to a human    comprising a TOI nucleotide sequence comprising an allele selected    as having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, whereby a    TOI encoded by said nucleotide sequence is targeted.-   37. The method of paragraph 36, wherein the method comprises    targeting a human TOI comprising an amino acid sequence with said    ligand to treat and/or prevent said disease or condition in said    human, wherein said amino acid sequence is encoded by a nucleotide    sequence comprising an allele having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%.-   38. A method of Target of Interest (TOI) genotyping a nucleic acid    sample of a human, the method comprising assaying in the sample the    presence of a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50%.-   39. A method of Target of Interest (TOI) typing a protein sample of    a human, the method comprising assaying the sample the presence of a    TOI amino acid sequence encoded by a TOI nucleotide sequence    comprising an allele having a cumulative human allele frequency of    less than 50% and/or having a total human genotype frequency of less    than 50%.-   40. The method of paragraph 38 or 39, comprising obtaining a sample    of serum, blood, faeces, hair, urine or saliva from a human, whereby    the nucleic acid or protein sample is obtained for use in the step    of assaying said sequence.-   41. The method of any one of paragraphs 38-40, comprising using a    ligand capable of targeting a nucleic acid sequence comprising an    allele having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50% or a    ligand capable of specifically binding the TOI encoded by said    nucleic acid sequence to carry out said identifying step.-   42. A diagnostic kit comprising a ligand that is capable of binding    a human Target of Interest (TOI) comprising an amino acid sequence    encoded by a TOI nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50% and instructions for    carrying out the method of any one of paragraphs 38-41.-   43. The diagnostic kit wherein the ligand is selected from an    antibody, antibody fragment, antibody portion, affybody,    oligonucleotide, modified oligonucleotide, antisense    oligonucleotide, siRNA, and microRNA.-   44. A diagnostic kit comprising a nucleic acid probe comprising a    nucleotide sequence that specifically hybridises a Target of    Interest (TOI) nucleotide sequence comprising an allele having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50% or an RNA transcript    thereof and instructions for carrying out the method of paragraph 38    or 39.-   45. The method, ligand, composition, kit or use of any preceding    paragraph, wherein the TOI is encoded by a nucleotide sequence    having a cumulative human allele frequency from 1 to 10% and/or a    total human genotype frequency from 1 to about 15% or from 1 to 15%.-   46. The method, ligand, composition, kit or use of any preceding    paragraph wherein the TOI is a human TOI selected from Table 5;    optionally for treating and/or preventing a corresponding disease or    condition as set out in Table 5.-   47. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising administering to the    human determined to be positive for the TOI polymorphic variant,    wherein the TOI in said human is encoded by a nucleotide sequence    comprising an allele having a cumulative human allele frequency of    less than 50% and/or wherein the TOI in said human is encoded by a    nucleotide sequence comprising an allele having total human genotype    frequency of less than 50% an anti-TOI ligand to target the TOI in    the human to treat or prevent said disease or condition.-   48. The method of paragraph 47, wherein the anti-TOI ligand is    selected from an antibody, an antibody portion, an antibody    fragment, an affibody, an antisense oligonucleotide, an siRNA, and a    microRNA.-   49. An anti-TOI ligand that specifically binds to a TOI as defined    in paragraph 1, for use in the method of any preceding paragraph    (eg, for use in the method of any of paragraphs 1 to 8 and 19 to    23).-   50. The ligand of paragraph 49, wherein the ligand is an anti-TOI    trap, antibody or antibody fragment.-   51. The ligand of paragraph 49 or 50, wherein the ligand is for    subcutaneous or intravenous administration to a human, or is    comprised by an injectable preparation.-   52. An injection device comprising the ligand of any one of    paragraphs 49 to 51.

Additional Tailoring of Ligands to Genotype and/or Phenotype of theHuman Patient

As described herein, the present invention contemplates ligands (eg,antibodies and fragments) whose binding site specificities have beenmatched to one or more variant human TOIs (eg, PCSK9 or IL6R).Additionally or alternatively (and as further illustrated in thenon-limiting Examples below), an optional aspect of the inventionprovides for matching of other features of the ligand to the patient'sgenotype or phenotype. In this respect, for example, the inventionincludes the ability to match amino acid sequence variation in a humanpatient to one or more ligand sequences or domains outside of thebinding sites. For example, where the ligand comprises or consists of ahuman TOI-binding antibody or an anti-human TOI receptor Fc fusion, thisaspect of the invention provides for more tailored matching of one ormore constant region domains (eg, the Fc) to the patient genotype orphenotype. Additionally or alternatively, it is contemplated thatsequence variation in the binding site can be similarly matched to thepatient's genotype or phenotype. The present inventor has done this byconsidering the SNP occurrences in sequences encoding one or more partsof the ligand, eg, SNP occurrences in one, more or all of the genesegments from which the variable domain(s) and/or constant regiondomain(s) are derived. The inventor realised that it would be desirableto match the ligand to one or more corresponding variant SNPs found inthe patient to be treated therapeutically and/or prophylactically.Matching could involve designing the ligand specifically for a patientof known phenotype and/or genotype, or matching could involve choosing aligand by determining that there is correspondence between variation inthe patient's phenotype or genotype with the variation in the ligandamino acid and/or corresponding nucleotides.

A key consideration for the inventor was the desire to promotecompatibility of the ligand with the patient's body, and in particular,the possible patient immune system responses to administered ligands.For example, it has been observed that human patients receiving human orhumanised antibody drugs may mount an immune response against theincoming antibody (a so-called HAHA response) which results in thepatient producing anti-drug antibodies as a result of the patient'simmune system recognising the drug as foreign. For example, studies havesuggested that some patients receiving HUMIRA™ (adalimumab), currentlythe biggest selling antibody medicine, mount a HAHA immune responseagainst the medicine, and this may impact treatment adversely. Referenceis made to JAMA. 2011; 305(14):1460-1468. doi:10.1001/jama.2011.406:“Development of Antidrug Antibodies Against Adalimumab and AssociationWith Disease Activity and Treatment Failure During Long-term Follow-up”,G M Bartelds et al. The authors concluded that results of this studyshowed that development of antidrug antibodies was associated with anegative outcome of adalimumab treatment in human RA patients. It wasreported that not only did patients with anti-adalimumab antibodiesdiscontinue treatment more often and earlier than patients withoutanti-adalimumab antibodies, they also had a higher disease activityduring treatment and only rarely came into remission. In addition,reportedly the data showed that two-thirds of the anti-adalimumabantibody-positive patients developed these antibodies in the first 28weeks of treatment and that the presence of anti-adalimumab antibodiessubstantially influenced serum adalimumab concentrations.

This HAHA theme is, therefore, a significant concern and this has beenconsidered by the regulatory authorities. For example, the EuropeanMedicines Agency (EMA) has issued a “Guideline on immunogenicityassessment of monoclonal antibodies intended for in vivo clinical use”(available on the world wide web atema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/06/WC500128688.pdf;EMA/CHMP/BMWP/86289/2010, addendum to EMEA/CHMP/BMWP/14327/2006), whichcame into force on 1^(st) December 2012. As such, it is good practicefor researchers to identify and assess risk of anti-antibody drugoccurrence and effects.

The present aspect of the invention, by more closely tailoring theligand itself (as well as its specificity) to the patient, helps toaddress these considerations when designing and administering anti-humanTOI medicines for treating and/or preventing human TOI-related diseasesand conditions.

The inventor also considered the desirability to tailor the variation inthe ligand constant region (eg, for an antibody or Fc-containingligand), mindful that then the constant region being administered to thepatient would be tuned to the various components, such as patient's Fcreceptors, that would interact with the constant region in the patient.Good Fc/Fc receptor interactions can be important for drug recycling(via the FcRn) to provide for useful half-lives in vivo or for use incell killing, eg, for cancer indications. In this way it is possible,therefore, to tune the effector function of the constant region (eg, Fc)to the patient more closely, to promote efficacy. For example, moreefficacious drugs are desirable for better patient treatment and mayprovide the possibility of lowered dosing and/or dosing frequencies.

Thus, in examples of this aspect, the invention provides the following(set out as clauses):—

-   1. The ligand, method, use, kit or composition of the invention,    wherein    -   (i) the ligand (eg, antibody or fragment) comprises        -   (c) a variable domain that is encoded by a human V region            nucleotide sequence, wherein the V nucleotide sequence is            derived from recombination of human VH, D and JH gene            segments or human VL and JL gene segments; or        -   (d) a constant region domain encoded by a C region gene            segment;        -   Wherein a first gene segment of said gene segments of (a),            or said C region gene segment of (b) comprises a first            single nucleotide polymorphism (SNP) encoding a first amino            acid polymorphism; and        -   (ii) the genome of said human comprises said first SNP or            wherein said human expresses (a′) an antibody variable            domain comprising said first amino acid polymorphism or (b′)            an antibody constant domain comprising said first amino acid            polymorphism.    -   2. The ligand, method, use, kit or composition of clause 1,        wherein blood of said human comprises substantially no        antibodies that specifically bind to the domain comprising said        first amino acid polymorphism as determined in an in vitro        binding assay.    -   3. The ligand, method, use, kit or composition of clause 2,        wherein SPR is used to carry out said assay.        -   In an alternative, ELISA is used.    -   4. The ligand, method, use, kit or composition of any one of        clauses 1 to 3, wherein the genome of said human comprises said        first gene segment (when (a) applies) or said C region gene        segment (when (b) applies).    -   5. The ligand, method, use, kit or composition of any one of        clauses 1 to 4, wherein said first segment or a second segment        of said segments of (a), or said C region gene segment of (b),        comprises a second SNP encoding a second amino acid        polymorphism; and wherein the genome of said human comprises        said second SNP or wherein said human expresses (a″) an antibody        variable domain comprising said second amino acid polymorphism        or (b″) an antibody constant region domain comprising said first        and second amino acid polymorphisms.    -   6. The ligand, method, use, kit or composition of clause 5,        wherein said human expresses an antibody variable domain        comprising said first and second amino acid polymorphisms.-   7. The ligand, method, use, kit or composition of clause 5 or 6,    wherein the first and second SNPs of said genome are comprised by    the same antibody gene segment.    -   For example, the first and second SNPs of the genome are        comprised by an IGHG1*01 gene segment and said first segment        of (a) is an IGHG1*01 gene segment.    -   For example, the first and second SNPs of the genome are        comprised by an IGHG2*01 gene segment and said first segment        of (a) is an IGHG2*01 gene segment.-   8. The ligand, method, use, kit or composition of any one of clauses    1 to 7, wherein each SNP is a variable region gene segment SNP.-   9. The ligand, method, use, kit or composition of any one of clauses    1 to 7, wherein each SNP is a constant region gene segment SNP, eg    each SNP is a gamma-1 constant region gene segment SNP, or a gamma-2    constant region gene segment SNP, or a gamma-3 constant region gene    segment SNP or a gamma-4 constant region gene segment SNP.-   10. The ligand, method, use, kit or composition of clause 9, wherein    the first SNP is a CH1, CH2, CH3 or CH4 gene segment SNP and/or the    second SNP is a CH1, CH2, CH3 or CH4 gene segment SNP.-   11. The ligand, method, use, kit or composition of any one of    clauses 1 to 8, wherein each SNP is a variable domain SNP, eg, a VH    domain SNP, or a Vκ domain SNP, or a Vλ SNP.-   12. The ligand, method, use, kit or composition of any one of    clauses 1 to 11, wherein said constant region domain of (b) is    comprised by an antibody Fc region.-   13. The ligand, method, use, kit or composition of any one of    clauses 1 to 12, wherein the ligand (eg, antibody or fragment) has    been determined to specifically bind one or more human TOI variants    as disclosed herein, for example, with a KD of 1 nM or less (eg, 100    or 10 pM or less) as determined by SPR.-   14. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 13), wherein the ligand    comprises or consists of an antibody or fragment that comprises a    human antibody variable domain derived from the recombination of a    human V gene segment and a human J gene segment (and optionally a    human D gene segment when the variable domains are VH domains); and    wherein the genome of the human comprises said human V gene segment    and/or the human expresses antibodies comprising antibody variable    domains derived from the recombination of said human V gene segment    and a human J gene segment (and optionally a human D gene segment).    -   In an example, the V gene segment is any of the V gene segments        disclosed in WO2013041844, a 1000 Genomes database and/or        www.imgt.org, the disclosures of which (including disclosure        relating to sequence) is explicitly incorporated herein by        reference for use in the present invention.-   15. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 14), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human heavy chain constant    domain encoded by a first constant region nucleotide sequence; and    wherein the genome of the human comprises a heavy chain constant    region nucleotide sequence that is identical to said first constant    region nucleotide sequence and/or the human expresses antibodies    comprising said human constant domain.-   16. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 15), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH1    domain encoded by a CH1 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH1 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH1 domain.-   17. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 16), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH2    domain encoded by a CH2 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH2 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH2 domain.-   18. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 17), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH3    domain encoded by a CH3 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH3 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH3 domain.-   19. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 18), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain CH4    domain encoded by a CH4 nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said CH4 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH4 domain.-   20. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 19), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused human TOI receptor) comprises a human gamma heavy chain Fc    region encoded by a Fc nucleotide sequence; and wherein the genome    of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said Fc nucleotide sequence    and/or the human expresses antibodies comprising said human gamma Fc    region.-   21. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein said human gamma heavy chain is a human    gamma-1 heavy chain-   22. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein said human gamma heavy chain is a human    gamma-2 heavy chain-   23. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein ligand comprises a human IGHG1*01 gamma-1    heavy chain constant region.-   24. The ligand, method, use, kit or composition of any one of    clauses 16 to 20, wherein ligand comprises a human IGHG2*01 gamma-1    heavy chain constant region.-   25. The ligand, method, use, kit or composition of any one of    clauses 15 to 24, wherein the human has been or is genotyped as    positive for said heavy chain constant region nucleotide sequence.-   26. The ligand, method, use, kit or composition of clause 23,    wherein the human has been or is genotyped as positive for human    IGHG1*01 nucleotide sequence.-   27. The ligand, method, use, kit or composition of clause 24,    wherein the human has been or is genotyped as positive for human    IGHG2*01 nucleotide sequence.-   28. The ligand, method, use, kit or composition of any one of    clauses 16 to 24, wherein the human has been or is phenotyped as    positive for said gamma heavy chain constant domain, CH1, CH2, CH3,    CH4 or Fc.-   29. The ligand, method, use, kit or composition of clause 28, (i)    when dependent from clause 23, wherein the human has been or is    phenotyped as positive for a human IGHG1*01 gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc or (ii) when dependent    from clause 24, wherein the human has been phenotyped as positive    for a human IGHG2*01 gamma heavy chain constant domain, CH1, CH2,    CH3, CH4 or Fc.-   30. The method or use of any one of clauses 16 to 24 and 26 to 29,    comprising genotyping the human as positive for said gamma heavy    chain constant region nucleotide sequence, eg, positive for said    gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc    nucleotide sequence; positive for said human IGHG1*01 gamma heavy    chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence;    or positive for said human IGHG2*01 gamma heavy chain constant    region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence.-   31. The method or use of any one of clauses 16 to 24 and 26 to 30,    comprising phenotyping the human as positive for said gamma heavy    chain constant region, eg, positive for said gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc; positive for said human    IGHG1*01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or    Fc; or positive for said human IGHG2*01 gamma heavy chain constant    domain, CH1, CH2, CH3, CH4 or Fc.

Examples of Tailored Ligands

The inventor analysed amino acid variability and distribution amongstlarge representative human samples. The result of the analysis forexample antibody gene segments is shown in Table 9.

In a first example, the inventor identified the possibility ofaddressing the rarer IGH-gamma-1 SNPs 204D (observed cumulativefrequency of 0.296) and 206L (observed cumulative frequency of 0.283)individually or in combination. These residues are part of the CH3domain, and as such they form part of antibody Fc regions. Thus,matching of these CH3 variations with the patient is especiallybeneficial for reasons as discussed above. Thus, this example providesaspects set out in the following clauses.

-   32. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 31), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human gamma-1 heavy chain    constant region that comprises an Asp corresponding to position 204    of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID    NO: 42 and wherein the genome of the human comprises a gamma-1 heavy    chain constant region nucleotide sequence that encodes such an Asp    or Leu or the human expresses antibodies comprising human gamma-1    constant regions comprising such an Asp or Leu.    -   The skilled person will be familiar with techniques for        determining genome sequences of a human, eg, by using a sample        containing genomic DNA and/or RNA, sequencing and comparing        using bioinformatics or other computer tools to compare the        sampled sequence with sequences of human alleles (eg, as shown        in the IMGT, 100 genomes or other database as disclosed herein).        In an example, the sample is a blood or saliva or cheek swab        sample.-   33. The ligand, method, use, kit or composition of clause 32,wherein    the ligand comprises a human gamma-1 heavy chain constant region    that comprises an Asp corresponding to position 204 of SEQ ID NO: 42    and a Leu corresponding to position 206 of SEQ ID NO: 42.-   34. The ligand, method, use, kit or composition of clause 32 or    33,wherein the genome of the human comprises a gamma-1 heavy chain    constant region nucleotide sequence that encodes such an Asp and Leu    or the human expresses antibodies comprising human gamma-1 constant    regions comprising such an Asp and Leu.-   35. The ligand, method, use, kit or composition of clause 32, 33 or    34, wherein the ligand comprises a human IGHG1*01 gamma-1 heavy    chain constant region, eg, an Fc, CH1, CH2 and/or CH3 domain encoded    by human IGHG1*01.-   36. The ligand, method, use, kit or composition of any one of    clauses 32 to 35, wherein the genome of the human comprises a human    IGHG1*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG1*01    nucleotide sequence.-   37. The ligand, method, use, kit or composition of any one of    clauses 32 to 36, wherein the ligand comprises a hinge region    encoded by human IGHG1*01.-   38. The ligand, method, use, kit or composition of any one of    clauses 32 to 37, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 61.-   39. The ligand, method, use, kit or composition of any one of    clauses 32 to 38, wherein the human is of European ancestry.    -   As shown in Table 9, 204D and 206L are found in such humans.-   40. The ligand, method, use, kit or composition of any one of    clauses 32 to 39, wherein the human has been or is genotyped as    positive for said Asp and/or Leu.-   41. The ligand, method, use, kit or composition of any one of    clauses 32 to 40, wherein the human has been or is genotyped as    positive for human IGHG1*01.-   42. The ligand, method, use, kit or composition of any one of    clauses 32 to 41, wherein the human has been or is phenotyped as    positive for a human IGHG1*01 CH3.-   43. The method or use of any one of clauses 32 to 42, comprising    selecting a said human whose genome comprises a codon(s) encoding    said Asp and/or Leu; comprises human IGHG1*01; or comprises a human    IGHG1*01 CH3.-   44. The method or use of any one of clauses 32 to 43, comprising    selecting a said human whose phenotype comprises said Asp and/or    Leu; a human IGHG1*01 region; or a human IGHG1*01 CH3.-   44a. The ligand, method, use, kit or composition of any one of    clauses 32 to 44, wherein the human expresses antibodies comprising    human gamma-1 constant regions comprising such an Asp and Leu.

In a second example, the inventor identified the possibility ofaddressing IGH-gamma-2 SNPs. This included consideration of Fc regionvariation—in this respect, the inventor focused on positions 161 and 257which are in the Fc region. Thus, this example provides aspects set outin the following clauses.

-   45. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 31), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human gamma-2 heavy chain    constant region that comprises an amino acid selected from the group    consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,    an Asn corresponding to position 75 of SEQ ID NO: 44, a Phe    corresponding to position 76 of SEQ ID NO: 44, a Val corresponding    to position 161 of SEQ ID NO: 44 and an Ala corresponding to    position 257 of SEQ ID NO: 44; and wherein the genome of the human    comprises a gamma-2 heavy chain constant region nucleotide sequence    that encodes such a selected amino acid or the human expresses    antibodies comprising human gamma-2 constant regions comprising such    a selected amino acid.-   46. The ligand, method, use, kit or composition of clause 45,    wherein the ligand comprises a human gamma-2 heavy chain constant    region that comprises (i) a Pro corresponding to position 72 of SEQ    ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a    Phe corresponding to position 76 of SEQ ID NO: 44 and    optionally (ii) a Val corresponding to position 161 of SEQ ID NO: 44    and/or an Ala corresponding to position 257 of SEQ ID NO: 44; and    wherein the genome of the human comprises a gamma-2 heavy chain    constant region nucleotide sequence that encodes such amino acids    of (i) or the human expresses antibodies comprising human gamma-2    constant regions comprising such amino acids of (i).    -   This example focuses on CH1 variation.-   47. The ligand, method, use, kit or composition of clause 45 or 46,    wherein the ligand comprises a human gamma-2 heavy chain constant    region that comprises (i) a Val corresponding to position 161 of SEQ    ID NO: 44 and an Ala corresponding to position 257 of SEQ ID NO: 44    and optionally (ii) an amino acid selected from the group consisting    of a Pro corresponding to position 72 of SEQ ID NO: 44, an Asn    corresponding to position 75 of SEQ ID NO: 44 and a Phe    corresponding to position 76 of SEQ ID NO: 44; and wherein the    genome of the human comprises a gamma-2 heavy chain constant region    nucleotide sequence that encodes such amino acids of (i) or the    human expresses antibodies comprising human gamma-2 constant regions    comprising such amino acids of (i).    -   This example focuses on Fc variation.-   48. The ligand, method, use, kit or composition of any one of    clauses 45 to 47, wherein the ligand comprises a human IGHG2*01    gamma-2 heavy chain constant region, eg, an Fc, CH1, CH2 and/or CH3    domain encoded by human IGHG2*01.-   49. The ligand, method, use, kit or composition of any one of    clauses 45 to 48, wherein the genome of the human comprises a human    IGHG2*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG2*01    nucleotide sequence.-   50. The ligand, method, use, kit or composition of any one of    clauses 45 to 49, wherein the ligand comprises a hinge region    encoded by human IGHG2*01.-   51. The ligand, method, use, kit or composition of any one of    clauses 45 to 50, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 63 or 65.-   52. The ligand, method, use, kit or composition of any one of    clauses 45 to 51, wherein the human is of European, African    American, or European American ancestry.-   53. The ligand, method, use, kit or composition of any one of    clauses 45 to 52, wherein the human has been or is genotyped as    positive for one, more or all of said Pro, Asn, Phe, Val and Ala.-   54. The ligand, method, use, kit or composition of any one of    clauses 45 to 53, wherein the human has been or is genotyped as    positive for human IGHG2*01.-   55. The ligand, method, use, kit or composition of any one of    clauses 45 to 54, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH1.-   56. The ligand, method, use, kit or composition of any one of    clauses 45 to 55, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH2.-   57. The ligand, method, use, kit or composition of any one of    clauses 45 to 56, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH3.-   58. The method or use of any one of clauses 45 to 57, comprising    selecting a said human whose genome comprises a codon(s) encoding    one, more or all of said Pro, Asn, Phe, Val and Ala; comprises human    IGHG2*01; or comprises a human IGHG2*01 CH1, CH2 and/or CH3.-   59. The method or use of any one of clauses 45 to 58, comprising    selecting a said human whose phenotype comprises one, more or all of    said Pro, Asn, Phe, Val and Ala; a human IGHG2*01 region; or a human    IGHG2*01 CH1, CH2 and/or CH3.-   60. The ligand, method, use, kit or composition of any one of    clauses 45 to 59, wherein the human expresses antibodies comprising    human gamma-2 constant regions comprising such a Pro, Asn, Phe, Val    and Ala.

In a third example, the inventor addressed human kappa constant regionvariation. Thus, the present aspect of the invention also provides thefollowing.

-   61. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 60), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; and    wherein the genome of the human comprises a kappa light chain    constant region nucleotide sequence that encodes such a Val or Cys    or the human expresses antibodies comprising human kappa light chain    constant regions comprising such a Val or Cys.-   62. The ligand, method, use, kit or composition of clause 61,    wherein the ligand comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50.-   63. The ligand, method, use, kit or composition of clause 61 or 62,    wherein the genome of the human comprises a kappa light chain    constant region nucleotide sequence that encodes such a Val and Cys    or the human expresses antibodies comprising human kappa constant    regions comprising such a Val and Cys.-   64. The ligand, method, use, kit or composition of any one of    clauses 61 to 63, wherein the antibody or fragment comprises a human    IGKC*01 kappa light chain constant region.-   65. The ligand, method, use, kit or composition of any one of    clauses 61 to 64, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises light chains that comprise    SEQ ID NO: 62 or 66.-   66. The ligand, method, use, kit or composition of any one of    clauses 61 to 65, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises a light chain variable    domain derived from recombination of a human Vκ gene segment and a    human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ    ID NO: 57).-   67. The ligand, method, use, kit or composition of any one of    clauses 61 to 66, wherein the human has been or is phenotyped as    positive for said Val and/or Cys.-   68. The ligand, method, use, kit or composition of any one of    clauses 61 to 67, wherein the human has been or is genotyped as    positive for human IGKC*01.-   69. The ligand, method, use, kit or composition of any one of    clauses 61 to 68, wherein the human has been or is phenotyped as    positive for a human IGKC*01 domain.-   70. The method or use of any one of clauses 61 to 69, comprising    selecting a said human whose genome comprises a codon(s) encoding    said Val and/or Cys; or comprises human IGKC*01.-   71. The method or use of any one of clauses 61 to 70, comprising    selecting a said human whose phenotype comprises such a Val and/or    Cys; or comprises a human IGKC*01 domain.-   72. The ligand, method, use, kit or composition of any one of    clauses 61 to 71, wherein the human expresses antibodies comprising    human kappa constant domains comprising such a Val and Cys, eg,    expresses human IGKC*01 constant domains.

In a fourth example, the inventor addressed human lambda constant regionvariation. Thus, this example provides aspects set out in the followingclauses.

-   73. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 60), wherein the ligand    (eg, comprising or consisting of an antibody or fragment or an    Fc-fused TOI receptor) comprises a human IGLC2*01 light chain    constant region; and wherein the genome of the human comprises a    human IGLC2*01 nucleotide sequence or the human expresses antibodies    comprising human light chain IGLC2*01 constant regions.-   74. The ligand, method, use, kit or composition of clause 73,    wherein the antibody comprises light chains that comprise SEQ ID NO:    64.-   75. The ligand, method, use, kit or composition of clause 73 or 74,    wherein the human has been or is genotyped as positive for human    IGLC2*01.-   76. The ligand, method, use, kit or composition of any one of    clauses 73 to 75, wherein the human has been or is phenotyped as    positive for a human IGLC2*01 domain.-   77. The method or use of any one of clauses 73 to 76, comprising    selecting a said human whose genome comprises human IGLC2*01.-   78. The method or use of any one of clauses 73 to 77, comprising    selecting a said human whose phenotype comprises a human IGLC2*01    domain.-   79. The ligand, method, use, kit or composition of any one of    clauses 73 to 78, wherein the human expresses antibodies comprising    human lambda IGLC2*01 constant domains.

In a fifth example, the inventor addressed human heavy chain variableregion variation. Thus, this example provides aspects set out in thefollowing clauses.

-   80. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 79), wherein the ligand    comprises or consists of an antibody or fragment, wherein the    antibody or fragment comprises a VH domain that is derived from the    recombination of a human VH gene segment, a human D gene segment and    a human JH gene segment, wherein the VH gene segment is selected    from the group consisting of (i) IGHV1-18*01 and the genome of the    human comprises a human IGHV1-18*01 nucleotide sequence or the human    expresses antibodies comprising variable domains derived from the    recombination of human IGHV1-18*01; or (ii) IGVH1-46*01 and the    genome of the human comprises a human IGHV1-46*01 nucleotide    sequence or the human expresses antibodies comprising variable    domains derived from the recombination of human IGHV1-46*01.-   81. The ligand, method, use, kit or composition of clause 80,    wherein the antibody or fragment comprises a one more or all of a    CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human    IGHG2*01.-   82. The ligand, method, use, kit or composition of clause 80 or 81,    wherein the antibody or fragment comprises heavy chains that    comprise SEQ ID NO: 63 or 65.-   83. The ligand, method, use, kit or composition of any one of    clauses 80 to 82, wherein the human has been or is genotyped as    positive for said selected VH gene segment, positive for human    IGHV1-18*01 or IGVH1-46*01.-   84. The method or use of any of clauses 80 to 83, comprising    genotyping the human as positive for said selected VH gene segment,    eg, positive for human IGHV1-18*01 or IGVH1-46*01.

In a sixth example, the inventor addressed human light chain variableregion variation. Thus, this example provides aspects set out in thefollowing clauses.

-   85. The ligand, method, use, kit or composition of the invention    (eg, according to any one of clauses 1 to 84), wherein the ligand    comprises or consists of an antibody or fragment, wherein the    antibody or fragment comprises a VL domain that is derived from the    recombination of a human VL gene segment and a human JL gene    segment, wherein the VL gene segment is selected from the group    consisting of (i) IGKV4-1*01 and the genome of the human comprises a    human IGKV4-1*01 nucleotide sequence or the human expresses    antibodies comprising variable domains derived from the    recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome    of the human comprises a human IGLV2-14*01 nucleotide sequence or    the human expresses antibodies comprising variable domains derived    from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02    and the genome of the human comprises a human IGKV1-13*02 nucleotide    sequence or the human expresses antibodies comprising variable    domains derived from the recombination of human IGKV1-13*02.-   86. The ligand, method, use, kit or composition of clause 85,    wherein the antibody comprises light chains that comprise SEQ ID NO:    62, 64 or 66.-   87. The ligand, method, use, kit or composition of clause 85 or 86,    wherein the antibody or fragment comprises a light chain variable    domain derived from recombination of a human Vκ gene segment and a    human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ    ID NO: 57; wherein (i) or (iii) applies.-   88. The ligand, method, use, kit or composition of any one of    clauses 85 to 87, wherein the human has been or is genotyped as    positive for said selected VL gene segment, eg, positive for human    IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02.-   89. The method or use of clause 88, comprising genotyping the human    as positive for said selected

VL gene segment, eg, genotyping the human as positive for humanIGKV4-1*01, IGLV2-14*01 or IGKV1-13*02.

-   90. The ligand, method, use, kit or composition of any one of    clauses 1 to 89, wherein the ligand (eg, antibody or fragment) binds    said human TOI with a dissociation constant (Kd) of 1 nM or less as    determined by SPR, (eg, 100, 10 or 1 pM or less).

In a specific embodiment, the ligand, antibody or fragment of presentinvention comprises an Fc region, wherein the Fc region comprises atleast one non-native amino acid residue selected from the groupconsisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V,234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y,2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y,240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R. 243W, 243L 243Y,243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E,256M, 262I, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 264I, 264W,264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V,2651, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N,296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981, 298T,298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D,325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301,330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E,331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S,332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may compriseadditional and/or alternative non-native amino acid residues known toone skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

In an example the ligand, antibody or fragment comprises one or morehuman binding sites that specifically bind the TOI (eg, PCSK9, IL4Ra,IL6R, VEGFA or Nav1.7). In an embodiment, the binding sites comprise orconsist of human antibody variable domains or human receptors for theTOI (eg, Human receptor binding sites for VEGFA). Furthermore, theligand, antibody or fragment can be optionally fully human (eg,comprising human constant regions, eg, human Fc regions with or withouthuman CL regions). In an example, the ligand is aflibercept, alirocumab,sarilumab or dupilumab. Fully human ligands maximise compatibility withthe human patient when used in the context of the invention wherein theV and/or C regions are tailored to the human genotype and/or phenotypeas per the description herein.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is encoded by a        nucleotide sequence having a cumulative human allele frequency        of less than 50% and/or wherein the TOI in said human is encoded        by a nucleotide sequence having a total human genotype frequency        of less than 50%;        -   wherein    -   b. Before step (a) said human has been or is genotyped as        positive for said nucleotide sequence or phenotyped as positive        for said TOI variant.-   2. The method of paragraph 1, wherein before step (a) the ligand has    been or is determined as being capable of binding to said TOI    variant.-   3. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is encoded by a        nucleotide sequence having a cumulative human allele frequency        of less than 50% and/or wherein the TOI in said human is encoded        by a nucleotide sequence having a total human genotype frequency        of less than 50%;        -   wherein    -   b. Before step (a) the ligand has been or is determined as        capable of binding to said TOI variant.-   4. The method of paragraph 3, wherein the genome of said human    comprises a nucleotide sequence encoding said TOI variant; and    before step (a) said nucleotide sequence has been or is determined    as having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50%.-   5. The method of paragraph 3 or 4, wherein said human has been or is    genotyped as positive for said variant nucleotide sequence before    step (a).-   6. The method of any preceding paragraph, wherein the human has been    or is phenotyped as positive for said TOI variant before step (a).-   7. The method of any preceding paragraph, wherein said frequency is    less than 10 or 15%.-   8. The method of any preceding paragraph, wherein the ligand is    capable of binding to two or more different TOI variants, each being    encoded by a nucleotide sequence having a cumulative human allele    frequency of less than 50% and/or having a total human genotype    frequency of less than 50%.-   9. A method of treating or preventing a disease or condition in a    human, wherein the disease or condition is mediated by a Target of    Interest (TOI), wherein the TOI is present in humans as different    polymorphic variants, the method comprising    -   a. Administering to the human an anti-TOI ligand to target a TOI        variant in the human and treat or prevent said disease or        condition, wherein the TOI in said human is a variant encoded by        a nucleotide sequence having a cumulative human allele frequency        of more than 50% and/or having a total human genotype frequency        of more than 50%;        -   wherein    -   b. Before step (a) said human has been or is genotyped as        negative for a variant nucleotide sequence having a cumulative        human allele frequency of less than 50% and/or having a total        human genotype frequency of less than 50%; or phenotyped as        negative for a TOI variant encoded by a nucleotide sequence        having a cumulative human allele frequency of less than 50%        and/or having a total human genotype frequency of less than 50%.-   10. The method of paragraph 9, wherein before step (a), the human    has been or is phenotyped as positive for the most frequent TOI    variant or genotyped for the nucleotide sequence thereof-   11. The method of paragraph 9 or 10, wherein before step (a) the    ligand has been or is determined as being capable of binding to the    most frequent TOI variant.-   12. The method of paragraph 9, 10 or 11, wherein before step (a) the    ligand has been or is determined as being substantially incapable of    neutralising or inhibiting said TOI variant recited in step (b).-   13. The method of any one of paragraphs 9 to 12, wherein the ligand    is capable of binding to the most frequent TOI variant.-   14. The method of any one of paragraphs 9 to 13, wherein the ligand    is capable of binding to two or more different TOI variants, each    being encoded by a nucleotide sequence having a cumulative human    allele frequency of more than 50%.-   15. The method of any preceding paragraph, wherein said variant    nucleotide sequence recited in step (a) has been or is determined as    being present in at least 2 different human ethnic populations.-   16. The method of any preceding paragraph, wherein said human    frequency is the frequency in a database of naturally-occurring    sequences sampled from at least 15 different human ethnic    populations and comprising at least 1000 sequences.-   17. An anti-human TOI ligand for use in a method of treating and/or    preventing a TOI-mediated disease or condition in a human, wherein    the TOI is present in humans as different polymorphic variants and    wherein the genome of said human comprises a TOI nucleotide sequence    having a cumulative human allele frequency of less than 50% and/or a    total human genotype frequency of less than 50%, the method    comprising administering the ligand to the human.-   18. The ligand of paragraph 17, wherein the ligand has been or is    determined as capable of binding the human TOI encoded by said    nucleotide sequence.-   19. A ligand that binds a human TOI comprising an amino acid    sequence encoded by a TOI nucleotide sequence having a cumulative    human allele frequency of less than 50% and/or a total human    genotype frequency of less than 50%, for use in a method comprising    the step of using the ligand to target said TOI in a human to treat    and/or prevent a disease or condition mediated by TOI, the method    comprising administering the ligand to the human.-   20. The ligand of any one of paragraphs 17 to 19, wherein the human    has been or is genotyped as positive for said TOI nucleotide    sequence having a cumulative human allele frequency of less than    50%.    -   The ligand of any one of paragraphs 17 to 19, wherein the human        has been or is genotyped as positive for said TOI nucleotide        sequence having a total human genotype frequency of less than        50%.-   21. The ligand of any one of paragraphs 17 to 20, wherein the human    has been or is phenotyped as positive for a TOI encoded by a    nucleotide sequence having a cumulative human allele frequency of    less than 50% and/or having a total human genotype frequency of less    than 50%.-   22. The ligand of any one of paragraphs 17 to 21, wherein the human    has been or is genotyped as heterozygous for a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50%;    optionally wherein the human has been or is genotyped as comprising    a TOI nucleotide sequence having a cumulative human allele frequency    of less than 50% and a TOI nucleotide sequence having a cumulative    human allele frequency of more than 50% and/or having a total human    genotype frequency of more than 50%.-   23. The ligand of any one of paragraphs 17 to 22, wherein the genome    of the human has been or is genotyped as homozygous for a TOI    nucleotide sequence having a cumulative human allele frequency of    less than 50% and/or having a total human genotype frequency of less    than 50%.-   24. The ligand of any one of paragraphs 17 to 23, wherein the ligand    comprises an antibody binding site that binds a human TOI comprising    an amino acid sequence encoded by a TOI nucleotide sequence having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50%; and optionally has    been or is determined as capable of such binding.-   25. The ligand of paragraph 24, wherein the ligand is an antibody or    antibody fragment.-   26. The ligand of any one of paragraphs 17 to 23, wherein the ligand    comprises a nucleotide sequence that specifically hybridises to a    TOI nucleotide sequence having a cumulative human allele frequency    of less than 50% and/or having a total human genotype frequency of    less than 50% or an RNA transcript thereof; and/or the ligand    comprises a nucleotide sequence that comprises at least 10    contiguous nucleotides of a nucleotide sequence having a cumulative    human allele frequency of less than 50% and/or having a total human    genotype frequency of less than 50% or is an antisense sequence    thereof.-   27. The ligand of any one of paragraphs 17 to 26, wherein the genome    of said human comprises a nucleotide sequence having a cumulative    human allele frequency of less than 50% and the sequence is found in    at least 2 different ethnic populations.-   28. A pharmaceutical composition or kit for treating or preventing a    condition or disease mediated by a TOI as recited in any preceding    paragraph, the composition or kit comprising a ligand of any one of    paragraphs 17 to 27; and optionally in combination with a label or    instructions for use to treat and/or prevent said disease or    condition in a human; optionally wherein the label or instructions    comprise a marketing authorisation number (eg, an FDA or EMA    authorisation number); optionally wherein the kit comprises an    injection pen or IV container that comprises the ligand.-   29. A method of producing an anti-human TOI antibody binding site,    the method comprising obtaining a plurality of anti-TOI antibody    binding sites, screening the antibody binding sites for binding to a    TOI comprising an amino acid sequence encoded by a nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, or to a    peptide thereof that comprises an amino acid variation from the    corresponding sequence encoded by the TOI-encoding nucleotide    sequence having the highest cumulative human allele frequency and/or    the highest total human genotype frequency, and isolating an    antibody binding site that binds in the screening step.-   30. A method of producing an anti-human TOI antibody, the method    comprising immunising a non-human vertebrate (eg, a mouse or a rat)    with a TOI comprising an amino acid sequence encoded by a nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%, or to a    peptide thereof that comprises an amino acid variation from the    corresponding sequence encoded by the TOI-encoding nucleotide    sequence having the highest cumulative human allele frequency and/or    the highest total human genotype frequency, and isolating an    antibody that binds a TOI comprising an amino acid sequence encoded    by a TOI nucleotide sequence having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%, and optionally producing a TOI-binding fragment or    derivative of the isolated antibody.-   31. The method of paragraph 29 or 30, comprising the step of    obtaining a nucleic acid encoding the antibody, fragment, derivative    or binding site and optionally inserting the nucleic acid in an    expression vector.-   32. A kit for TOI genotyping a human, wherein the kit comprises a    nucleic acid comprising a nucleotide sequence that specifically    hybridises to a TOI nucleotide sequence selected having a cumulative    human allele frequency of less than 50% and/or a total human    genotype frequency of less than 50% or an RNA transcript thereof;    and/or the nucleic acid comprises a nucleotide sequence that    comprises at least 10 contiguous nucleotides of a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50% or is an    antisense sequence thereof-   33. A kit for TOI genotyping or phenotyping a human, wherein the kit    comprises a ligand according to any one of paragraphs 17 to 27 or an    antibody, fragment or derivative produced by the method of any one    of paragraphs 29 to 31.-   34. Use of an anti-TOI ligand that binds a human TOI comprising an    amino acid sequence encoded by a TOI nucleotide sequence having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50%, in the manufacture of a    medicament for treating and/or preventing a TOI-mediated disease or    condition in a human whose genome comprises a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or having a total human genotype frequency of less than 50%.-   35. Use of an anti-TOI ligand that binds a human TOI comprising an    amino acid sequence encoded by a TOI nucleotide sequence having a    cumulative human allele frequency of less than 50% and/or a total    human genotype frequency of less than 50%, in the manufacture of a    medicament for targeting said TOI in a human to treat and/or prevent    a disease or condition mediated by TOI.-   36. A method of targeting a TOI for treating and/or preventing a    TOI-mediated disease or condition in a human, the method comprising    administering an anti-TOI ligand to a human comprising a TOI    nucleotide sequence selected having a cumulative human allele    frequency of less than 50% and/or a total human genotype frequency    of less than 50%, whereby a TOI encoded by said nucleotide sequence    is targeted.-   37. The method of paragraph 36, wherein the method comprises    targeting a human TOI comprising an amino acid sequence with said    ligand to treat and/or prevent said disease or condition in said    human, wherein said amino acid sequence is encoded by a nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50%.-   38. A method of TOI genotyping a nucleic acid sample of a human, the    method comprising identifying in the sample the presence of a TOI    nucleotide sequence having a cumulative human allele frequency of    less than 50% and/or having a total human genotype frequency of less    than 50%.-   39. A method of TOI typing a protein sample of a human, the method    comprising identifying in the sample the presence of a TOI amino    acid sequence encoded by a TOI nucleotide sequence having a    cumulative human allele frequency of less than 50% and/or having a    total human genotype frequency of less than 50%.-   40. The method of paragraph 38 or 39, comprising obtaining a sample    of serum, blood, faeces, hair, urine or saliva from a human, whereby    the nucleic acid or protein sample is obtained for use in the step    of identifying said sequence.-   41. The method of any one of paragraphs 38 to 40, comprising using a    ligand according to any one of paragraphs 17 to 27 to carry out said    identifying step.-   42. A diagnostic kit comprising a ligand that is capable of binding    a human TOI comprising an amino acid sequence encoded by a TOI    nucleotide sequence having a cumulative human allele frequency of    less than 50% and/or a total human genotype frequency of less than    50% and instructions for carrying out the method of paragraph 38 or    39.-   43. A diagnostic kit comprising a nucleic acid probe comprising a    nucleotide sequence that specifically hybridises a TOI nucleotide    sequence having a cumulative human allele frequency of less than 50%    and/or a total human genotype frequency of less than 50% or an RNA    transcript thereof and instructions for carrying out the method of    paragraph 38 or 39.-   44. The method, ligand, composition, kit or use of any preceding    paragraph, wherein the TOI is encoded by a nucleotide sequence    having a cumulative human allele frequency from 1 to 10% and/or a    total human genotype frequency from 1 to about 15% or from 1 to 15%.-   45. The method, ligand, composition, kit or use of any preceding    paragraph wherein the TOI is a human TOI selected from Table 5;    optionally for treating and/or preventing a corresponding disease or    condition as set out in table 5.-   46. The ligand, method, use, kit or composition of any preceding    paragraph, wherein    -   (i) the ligand (eg, antibody or fragment) comprises        -   (e) a variable domain that is encoded by a human V region            nucleotide sequence, wherein the V nucleotide sequence is            derived from recombination of human VH, D and JH gene            segments or human VL and JL gene segments; or        -   (f) a constant region domain encoded by a C region gene            segment;        -   Wherein a first gene segment of said gene segments of (a),            or said C region gene segment of        -   (b) comprises a first single nucleotide polymorphism (SNP)            encoding a first amino acid polymorphism; and        -   (ii) the genome of said human comprises said first SNP or            wherein said human expresses (a′) an antibody variable            domain comprising said first amino acid polymorphism or (b′)            an antibody constant domain comprising said first amino acid            polymorphism.-   47. The ligand, method, use, kit or composition of paragraph 46,    wherein blood of said human comprises substantially no antibodies    that specifically bind to the domain comprising said first amino    acid polymorphism as determined in an in vitro binding assay.-   48. The ligand, method, use, kit or composition of paragraph 47,    wherein SPR is used to carry out said assay.-   49. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 48, wherein the genome of said human comprises said    first gene segment (when (a) applies) or said C region gene segment    (when (b) applies).-   50. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 49, wherein said first segment or a second segment    of said segments of (a), or said C region gene segment of (b),    comprises a second SNP encoding a second amino acid polymorphism;    and wherein the genome of said human comprises said second SNP or    wherein said human expresses (a″) an antibody variable domain    comprising said second amino acid polymorphism or (b″) an antibody    constant region domain comprising said first and second amino acid    polymorphisms.-   51. The ligand, method, use, kit or composition of paragraph 50,    wherein said human expresses an antibody variable domain comprising    said first and second amino acid polymorphisms.-   52. The ligand, method, use, kit or composition of paragraph 50 or    51, wherein the first and second

SNPs of said genome are comprised by the same antibody gene segment.

-   53. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 52, wherein each SNP is a variable region gene    segment SNP.-   54. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 52, wherein each SNP is a constant region gene    segment SNP, eg each SNP is a gamma-1 constant region gene segment    SNP, or a gamma-2 constant region gene segment SNP, or a gamma-3    constant region gene segment SNP or a gamma-4 constant region gene    segment SNP.-   55. The ligand, method, use, kit or composition of paragraph 54,    wherein the first SNP is a CH1, CH2, CH3 or CH4 gene segment SNP    and/or the second SNP is a CH1, CH2, CH3 or CH4 gene segment SNP.-   56. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 53, wherein each SNP is a variable domain SNP, eg,    a VH domain SNP, or a Vκ domain SNP, or a 1a SNP.-   57. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 56, wherein said constant region domain of (b) is    comprised by an antibody Fc region.-   58. The ligand, method, use, kit or composition of any one of    paragraphs 46 to 57, wherein the ligand (eg, antibody or fragment)    has been determined to specifically bind one or more human TOI    variants as disclosed herein, for example, with a KD of 1 nM or less    (eg, 100 or 10 pM or less) as determined by SPR.-   59. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 58), wherein    the ligand comprises or consists of an antibody or fragment that    comprises a human antibody variable domain derived from the    recombination of a human V gene segment and a human J gene segment    (and optionally a human D gene segment when the variable domains are    VH domains); and wherein the genome of the human comprises said    human V gene segment and/or the human expresses antibodies    comprising antibody variable domains derived from the recombination    of said human V gene segment and a human J gene segment (and    optionally a human D gene segment).-   60. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 59), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused human TOI receptor) comprises a human heavy chain    constant domain encoded by a first constant region nucleotide    sequence; and wherein the genome of the human comprises a heavy    chain constant region nucleotide sequence that is identical to said    first constant region nucleotide sequence and/or the human expresses    antibodies comprising said human constant domain.-   61. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 60), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused human TOI receptor) comprises a human gamma heavy    chain CH1 domain encoded by a CH1 nucleotide sequence; and wherein    the genome of the human comprises a gamma heavy chain constant    region nucleotide sequence that is identical to said CH1 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH1 domain.-   62. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 61), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused human TOI receptor) comprises a human gamma heavy    chain CH2 domain encoded by a CH2 nucleotide sequence; and wherein    the genome of the human comprises a gamma heavy chain constant    region nucleotide sequence that is identical to said CH2 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH2 domain.-   63. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 62),    wherein, wherein the ligand (eg, comprising or consisting of an    antibody or fragment or an Fc-fused human TOI receptor) comprises a    human gamma heavy chain CH3 domain encoded by a CH3 nucleotide    sequence; and wherein the genome of the human comprises a gamma    heavy chain constant region nucleotide sequence that is identical to    said CH3 nucleotide sequence and/or the human expresses antibodies    comprising said human gamma CH3 domain.-   64. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 63), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused human TOI receptor) comprises a human gamma heavy    chain CH4 domain encoded by a CH4 nucleotide sequence; and wherein    the genome of the human comprises a gamma heavy chain constant    region nucleotide sequence that is identical to said CH4 nucleotide    sequence and/or the human expresses antibodies comprising said human    gamma CH4 domain.-   65. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of paragraphs 46 to 64), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused human TOI receptor) comprises a human gamma heavy    chain Fc region encoded by a Fc nucleotide sequence; and wherein the    genome of the human comprises a gamma heavy chain constant region    nucleotide sequence that is identical to said Fc nucleotide sequence    and/or the human expresses antibodies comprising said human gamma Fc    region.-   66. The ligand, method, use, kit or composition of any one of    paragraphs 61 to 65, wherein said human gamma heavy chain is a human    gamma-1 heavy chain.-   67. The ligand, method, use, kit or composition of any one of    paragraphs 61 to 65, wherein said human gamma heavy chain is a human    gamma-2 heavy chain.-   68. The ligand, method, use, kit or composition of any one of    paragraphs 61 to 65, wherein ligand comprises a human IGHG1*01    gamma-1 heavy chain constant region.-   69. The ligand, method, use, kit or composition of any one of    paragraphs 61 to 65, wherein ligand comprises a human IGHG2*01    gamma-1 heavy chain constant region.-   70. The ligand, method, use, kit or composition of any one of    paragraphs 60 to 69, wherein the human has been or is genotyped as    positive for said heavy chain constant region nucleotide sequence.-   71. The ligand, method, use, kit or composition of paragraph 68,    wherein the human has been or is genotyped as positive for human    IGHG1*01 nucleotide sequence.-   72. The ligand, method, use, kit or composition of paragraph 69,    wherein the human has been or is genotyped as positive for human    IGHG2*01 nucleotide sequence.-   73. The ligand, method, use, kit or composition of any one of    paragraphs 61 to 69, wherein the human has been or is phenotyped as    positive for said gamma heavy chain constant domain, CH1, CH2, CH3,    CH4 or Fc.-   74. The ligand, method, use, kit or composition of paragraph 63, (i)    when dependent from clause 23, wherein the human has been or is    phenotyped as positive for a human IGHG1*01 gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc or (ii) when dependent    from clause 24, wherein the human has been phenotyped as positive    for a human IGHG2*01 gamma heavy chain constant domain, CH1, CH2,    CH3, CH4 or Fc.-   75. The method or use of any one of paragraphs 61 to 69 and 71 to    74, comprising genotyping the human as positive for said gamma heavy    chain constant region nucleotide sequence, eg, positive for said    gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc    nucleotide sequence; positive for said human IGHG1*01 gamma heavy    chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence;    or positive for said human IGHG2*01 gamma heavy chain constant    region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence.-   76. The method or use of any one of paragraphs 61 to 69 and 71 to    75, comprising phenotyping the human as positive for said gamma    heavy chain constant region, eg, positive for said gamma heavy chain    constant domain, CH1, CH2, CH3, CH4 or Fc; positive for said human    IGHG1*01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or    Fc; or positive for said human IGHG2*01 gamma heavy chain constant    domain, CH1, CH2, CH3, CH4 or Fc.-   77. The ligand, method, use, kit or composition of any preceding    paragraph (eg, according to any one of clauses 46 to 76), wherein    the ligand (eg, comprising or consisting of an antibody or fragment    or an Fc-fused TOI receptor) comprises a human gamma-1 heavy chain    constant region that comprises an Asp corresponding to position 204    of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID    NO: 42 and wherein the genome of the human comprises a gamma-1 heavy    chain constant region nucleotide sequence that encodes such an Asp    or Leu or the human expresses antibodies comprising human gamma-1    constant regions comprising such an Asp or Leu.-   78. The ligand, method, use, kit or composition of paragraph 77,    wherein the ligand comprises a human gamma-1 heavy chain constant    region that comprises an Asp corresponding to position 204 of SEQ ID    NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42.-   79. The ligand, method, use, kit or composition of paragraph 77 or    78, wherein the genome of the human comprises a gamma-1 heavy chain    constant region nucleotide sequence that encodes such an Asp and Leu    or the human expresses antibodies comprising human gamma-1 constant    regions comprising such an Asp and Leu.-   80. The ligand, method, use, kit or composition of paragraph 77, 78    or 79, wherein the ligand comprises a human IGHG1*01 gamma-1 heavy    chain constant region, eg, an Fc, CH1, CH2 and/or CH3 domain encoded    by human IGHG1*01.-   81. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 80, wherein the genome of the human comprises a    human IGHG1*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG1*01    nucleotide sequence.-   82. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 81, wherein the ligand comprises a hinge region    encoded by human IGHG1*01.-   83. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 82, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 61.-   84. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 83, wherein the human is of European ancestry.-   85. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 84, wherein the human has been or is genotyped as    positive for said Asp and/or Leu.-   86. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 85, wherein the human has been or is genotyped as    positive for human IGHG1*01.-   87. The ligand, method, use, kit or composition of any one of    paragraphs 77 to 86, wherein the human has been or is phenotyped as    positive for a human IGHG1*01 CH3.-   88. The method or use of any one of paragraphs 77 to 87, comprising    selecting a said human whose genome comprises a codon(s) encoding    said Asp and/or Leu; comprises human IGHG1*01; or comprises a human    IGHG1*01 CH3.-   89. The method or use of any one of paragraphs 77 to 88, comprising    selecting a said human whose phenotype comprises said Asp and/or    Leu; a human IGHG1*01 region; or a human IGHG1*01 CH3.-   90. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the ligand (eg, comprising or consisting of an    antibody or fragment or an Fc-fused TOI receptor) comprises a human    gamma-2 heavy chain constant region that comprises an amino acid    selected from the group consisting of a Pro corresponding to    position 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of    SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,    a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala    corresponding to position 257 of SEQ ID NO: 44; and wherein the    genome of the human comprises a gamma-2 heavy chain constant region    nucleotide sequence that encodes such a selected amino acid or the    human expresses antibodies comprising human gamma-2 constant regions    comprising such a selected amino acid.-   91. The ligand, method, use, kit or composition of paragraph 90,    wherein the ligand comprises a human gamma-2 heavy chain constant    region that comprises (i) a Pro corresponding to position 72 of SEQ    ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a    Phe corresponding to position 76 of SEQ ID NO: 44 and    optionally (ii) a Val corresponding to position 161 of SEQ ID NO: 44    and/or an Ala corresponding to position 257 of SEQ ID NO: 44; and    wherein the genome of the human comprises a gamma-2 heavy chain    constant region nucleotide sequence that encodes such amino acids    of (i) or the human expresses antibodies comprising human gamma-2    constant regions comprising such amino acids of (i).-   92. The ligand, method, use, kit or composition of paragraph 90 or    91, wherein the ligand comprises a human gamma-2 heavy chain    constant region that comprises (i) a Val corresponding to position    161 of SEQ ID NO: 44 and an Ala corresponding to position 257 of SEQ    ID NO: 44 and optionally (ii) an amino acid selected from the group    consisting of a Pro corresponding to position 72 of SEQ ID NO: 44,    an Asn corresponding to position 75 of SEQ ID NO: 44 and a Phe    corresponding to position 76 of SEQ ID NO: 44; and wherein the    genome of the human comprises a gamma-2 heavy chain constant region    nucleotide sequence that encodes such amino acids of (i) or the    human expresses antibodies comprising human gamma-2 constant regions    comprising such amino acids of (i).-   93. The ligand, method, use, kit or composition of any one of    paragraph 90 to 92, wherein the ligand comprises a human IGHG2*01    gamma-2 heavy chain constant region, eg, an Fc, CH1, CH2 and/or CH3    domain encoded by human IGHG2*01.-   94. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 93, wherein the genome of the human comprises a    human IGHG2*01 nucleotide sequence or the human expresses antibodies    comprising human constant domains encoded by a human IGHG2*01    nucleotide sequence.-   95. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 94, wherein the ligand comprises a hinge region    encoded by human IGHG2*01.-   96. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 95, wherein the ligand comprises or consists of an    antibody, wherein the antibody comprises heavy chains that comprise    SEQ ID NO: 63 or 65.-   97. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 96, wherein the human is of European, African    American, or European American ancestry.-   98. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 97, wherein the human has been or is genotyped as    positive for one, more or all of said Pro, Asn, Phe, Val and Ala.-   99. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 98, wherein the human has been or is genotyped as    positive for human IGHG2*01.-   100. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 99, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH1.-   101. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 100, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH2.-   102. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 101, wherein the human has been or is phenotyped as    positive for a human IGHG2*01 CH3.-   103. The method or use of any one of paragraphs 90 to 102,    comprising selecting a said human whose genome comprises a codon(s)    encoding one, more or all of said Pro, Asn, Phe, Val and Ala;    comprises human IGHG2*01; or comprises a human IGHG2*01 CH1, CH2    and/or CH3.-   104. The method or use of any one of paragraphs 90 to 103,    comprising selecting a said human whose phenotype comprises one,    more or all of said Pro, Asn, Phe, Val and Ala; a human IGHG2*01    region; or a human IGHG2*01 CH1, CH2 and/or CH3.-   105. The ligand, method, use, kit or composition of any one of    paragraphs 90 to 104, wherein the human expresses antibodies    comprising human gamma-2 constant regions comprising such a Pro,    Asn, Phe, Val and Ala.-   106. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the ligand (eg, comprising or consisting of an    antibody or fragment or an Fc-fused TOI receptor) comprises a human    kappa light chain constant region that comprises a Val corresponding    to position 84 of SEQ ID NO: 50 or a Cys corresponding to position    87 of SEQ ID NO: 50; and wherein the genome of the human comprises a    kappa light chain constant region nucleotide sequence that encodes    such a Val or Cys or the human expresses antibodies comprising human    kappa light chain constant regions comprising such a Val or Cys.-   107. The ligand, method, use, kit or composition of paragraph 106,    wherein the ligand comprises a human kappa light chain constant    region that comprises a Val corresponding to position 84 of SEQ ID    NO: 50 and a Cys corresponding to position 87 of SEQ ID NO: 50.-   108. The ligand, method, use, kit or composition of paragraph 106 or    107, wherein the genome of the human comprises a kappa light chain    constant region nucleotide sequence that encodes such a Val and Cys    or the human expresses antibodies comprising human kappa constant    regions comprising such a Val and Cys.-   109. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 108, wherein the antibody or fragment comprises a    human IGKC*01 kappa light chain constant region.-   110. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 109, wherein the ligand comprises or consists of    an antibody, wherein the antibody comprises light chains that    comprise SEQ ID NO: 62 or 66.-   111. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 110, wherein the ligand comprises or consists of    an antibody, wherein the antibody comprises a light chain variable    domain derived from recombination of a human Vκ gene segment and a    human Jκ gene segment, wherein the Jκ gene segment is IGKJ2*01 (SEQ    ID NO: 57).-   112. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 111, wherein the human has been or is phenotyped    as positive for said Val and/or Cys.-   113. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 112, wherein the human has been or is genotyped as    positive for human IGKC*01.-   114. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 113, wherein the human has been or is phenotyped    as positive for a human IGKC*01 domain.-   115. The method or use of any one of paragraphs 106 to 114,    comprising selecting a said human whose genome comprises a codon(s)    encoding said Val and/or Cys; or comprises human IGKC*01.-   116. The method or use of any one of paragraphs 106 to 115,    comprising selecting a said human whose phenotype comprises such a    Val and/or Cys; or comprises a human IGKC*01 domain.-   117. The ligand, method, use, kit or composition of any one of    paragraphs 106 to 116, wherein the human expresses antibodies    comprising human kappa constant domains comprising such a Val and    Cys, eg, expresses human IGKC*01 constant domains.-   118. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the ligand (eg, comprising or consisting of an    antibody or fragment or an Fc-fused TOI receptor) comprises a human    IGLC2*01 light chain constant region; and wherein the genome of the    human comprises a human IGLC2*01 nucleotide sequence or the human    expresses antibodies comprising human light chain IGLC2*01 constant    regions.-   119. The ligand, method, use, kit or composition of paragraph 118,    wherein the antibody comprises light chains that comprise SEQ ID NO:    64.-   120. The ligand, method, use, kit or composition of paragraph 118 or    119, wherein the human has been or is genotyped as positive for    human IGLC2*01.-   121. The ligand, method, use, kit or composition of any one of    paragraphs 118 to 120, wherein the human has been or is phenotyped    as positive for a human IGLC2*01 domain.-   122. The method or use of any one of paragraphs 118 to 212,    comprising selecting a said human whose genome comprises human    IGLC2*01.-   123. The method or use of any one of clauses 73 to 77, comprising    selecting a said human whose phenotype comprises a human IGLC2*01    domain.-   124. The ligand, method, use, kit or composition of any one of    paragraphs 108 to 123, wherein the human expresses antibodies    comprising human lambda IGLC2*01 constant domains.-   125. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the ligand comprises or consists of an antibody    or fragment, wherein the antibody or fragment comprises a VH domain    that is derived from the recombination of a human VH gene segment, a    human D gene segment and a human JH gene segment, wherein the VH    gene segment is selected from the group consisting of (i)    IGHV1-18*01 and the genome of the human comprises a human    IGHV1-18*01 nucleotide sequence or the human expresses antibodies    comprising variable domains derived from the recombination of human    IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the human    comprises a human IGHV1-46*01 nucleotide sequence or the human    expresses antibodies comprising variable domains derived from the    recombination of human IGHV1-46*01.-   126. The ligand, method, use, kit or composition of paragraph 125,    wherein the antibody or fragment comprises a one more or all of a    CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human    IGHG2*01.-   127. The ligand, method, use, kit or composition of paragraph 125 or    126, wherein the antibody or fragment comprises heavy chains that    comprise SEQ ID NO: 63 or 65.-   128. The ligand, method, use, kit or composition of any one of    paragraphs 125 to 127, wherein the human has been or is genotyped as    positive for said selected VH gene segment, positive for human    IGHV1-18*01 or IGVH1-46*01.-   129. The method or use of any of paragraphs 125 to 128, comprising    genotyping the human as positive for said selected VH gene segment,    eg, positive for human IGHV1-18*01 or IGVH1-46*01.-   130. The ligand, method, use, kit or composition any preceding    paragraph, wherein the ligand comprises or consists of an antibody    or fragment, wherein the antibody or fragment comprises a VL domain    that is derived from the recombination of a human VL gene segment    and a human JL gene segment, wherein the VL gene segment is selected    from the group consisting of (i) IGKV4-1*01 and the genome of the    human comprises a human IGKV4-1*01 nucleotide sequence or the human    expresses antibodies comprising variable domains derived from the    recombination of human IGKV4-1*01; (ii) IGLV2-14*01 and the genome    of the human comprises a human IGLV2-14*01 nucleotide sequence or    the human expresses antibodies comprising variable domains derived    from the recombination of human IGLV2-14*01; or (iii) IGKV1-13*02    and the genome of the human comprises a human IGKV1-13*02 nucleotide    sequence or the human expresses antibodies comprising variable    domains derived from the recombination of human IGKV1-13*02.-   131. The ligand, method, use, kit or composition of paragraph 130,    wherein the antibody comprises light chains that comprise SEQ ID NO:    62, 64 or 66.-   132. The ligand, method, use, kit or composition of paragraph 130 or    131, wherein the antibody or fragment comprises a light chain    variable domain derived from recombination of a human Vκ gene    segment and a human Jκ gene segment, wherein the Jκ gene segment is    IGKJ2*01 (SEQ ID NO: 57; wherein (i) or (iii) applies.-   133. The ligand, method, use, kit or composition of any one of    paragraphs 130 to 132, wherein the human has been or is genotyped as    positive for said selected VL gene segment, eg, positive for human    IGKV4-1*01, IGLV2-14*01 or IGKV1-13*02.-   134. The method or use of paragraph 133, comprising genotyping the    human as positive for said selected VL gene segment, eg, genotyping    the human as positive for human IGKV4-1*01, IGLV2-14*01 or    IGKV1-13*02.-   135. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the ligand (eg, antibody or fragment) binds said    human TOI with a dissociation constant (Kd) of 1 nM or less as    determined by SPR, (eg, 100, 10 or 1 pM or less).-   136. The ligand, method, use, kit or composition of any preceding    paragraph, wherein the TOI is human PCSK9 or human IL-6R.

EXAMPLES Example 1 Rare PCSK9 Variants

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah andPrat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2cells lowers the levels of cell surface LDLR (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004).Experiments with mice have shown that increasing PCSK9 protein levelsdecreases levels of LDLR protein in the liver (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004), whilePCSK9 knockout mice have increased levels of LDLR in the liver (Rashidet al., 2005). Additionally, various human PCSK9 mutations that resultin either increased or decreased levels of plasma LDL have beenidentified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has beenshown to directly interact with the LDLR protein, be endocytosed alongwith the LDLR, and co-immunofluoresce with the LDLR throughout theendosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8)family of serine proteases (Seidah et al., 2003). Humans have nineprohormone-proprotein convertases that can be divided between the S8Aand S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4,PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B.Crystal and NMR structures of different domains from mouse furin and PC1reveal subtilisin-like pro- and catalytic domains, and a P domaindirectly C-terminal to the catalytic domain (Henrich et al., 2003;Tangrea et al., 2002). Based on the amino acid sequence similaritywithin this subfamily, all seven members are predicted to have similarstructures (Henrich et al., 2005). SKI-1/S1P and PCSK9 are classified insubfamily S8A. Sequence comparisons with these proteins also suggest thepresence of subtilisin-like pro- and catalytic domains (Sakai et al.,1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins theamino acid sequence C-terminal to the catalytic domain is more variableand does not suggest the presence of a P domain.

Prohormone-proprotein convertases are expressed as zymogens and theymature through a multi step process. The function of the pro-domain inthis process is two-fold. The pro-domain first acts as a chaperone andis required for proper folding of the catalytic domain (Ikemura et al.,1987). Once the catalytic domain is folded, autocatalysis occurs betweenthe pro-domain and catalytic domain. Following this initial cleavagereaction, the pro-domain remains bound to the catalytic domain where itthen acts as an inhibitor of catalytic activity (Fu et al., 2000). Whenconditions are correct, maturation proceeds with a second autocatalyticevent at a site within the pro-domain (Anderson et al., 1997). Afterthis second cleavage event occurs the pro-domain and catalytic domaindissociate, giving rise to an active protease.

Autocatalysis of the PCSK9 zymogen occurs between G1n152 and Ser153(VFAQ1SIP (SEQ ID NO: 116)) (Naureckiene et al., 2003), and has beenshown to be required for its secretion from cells (Seidah et al., 2003).A second autocatalytic event at a site within PCSK9's pro-domain has notbeen observed. Purified PCSK9 is made up of two species that can beseparated by non-reducing SDS-PAGE; the pro-domain at 17 Kd, and thecatalytic plus C-terminal domains at 65 Kd. PCSK9 has not been isolatedwithout its inhibitory pro-domain, and measurements of PCSK9's catalyticactivity have been variable (Naureckiene et al., 2003; Seidah et al.,2003).

In certain embodiments, a PCSK9 polypeptide includes terminal residues,such as, but not limited to, leader sequence residues, targetingresidues, amino terminal methionine residues, lysine residues, tagresidues and/or fusion protein residues. “PCSK9” has also been referredto as FH3, NARC1, HCHOLA3, proprotein convertase subtilisin/kexin type9, and neural apoptosis regulated convertase 1. The PCSK9 gene encodes aproprotein convertase protein that belongs to the proteinase K subfamilyof the secretory subtilase family. The term “PCSK9” denotes both theproprotein and the product generated following autocatalysis of theproprotein. When only the autocatalyzed product is being referred to(such as for an antigen binding protein or ligand that binds to thecleaved PCSK9), the protein can be referred to as the “mature,”“cleaved”, “processed” or “active” PCSK9. When only the inactive form isbeing referred to, the protein can be referred to as the “inactive”,“pro-form”, or “unprocessed” form of PCSK9. The term PCSK9 alsoencompasses PCSK9 molecules incorporating post-translationalmodifications of the PCSK9 amino acid sequence, such as PCSK9 sequencesthat have been glycosylated, PCSK9 sequences from which its signalsequence has been cleaved, PCSK9 sequence from which its pro domain hasbeen cleaved from the catalytic domain but not separated from thecatalytic domain (see, e.g., FIGS. 1A and 1B of US20120093818A1; whichis incorporated by reference herein in its entirety).

The present invention provides anti-PCSK9 ligands; and PCSK9-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PCSK9, in particular human PCSK9 or its ligands and inscreening assays to identify other antagonists of PCSK9 activity. Someof the ligands of the invention are useful for inhibiting binding ofPCSK9 to LDLR, or inhibiting PCSK9-mediated activities.

Anti-PCSK9 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human PCSK9, which is a commonly-occurring form (see, eg,US20120093818A1 and US20110065902A1; each of which is incorporated byreference herein in its entirety). While such therapies are useful forhuman patients harbouring this form of human PCSK9, the inventorconsidered it useful to investigate the possibility of targeting muchrarer but still naturally-occurring—forms of PCSK9 amongst humanpopulations. In this way, the inventor arrived at insight into thenatural occurrences and distributions of rarer human PCSK9 forms thatcan serve as useful targets (at the protein or nucleic acid level) forhuman treatment, prophylaxis and diagnosis pertinent to diseases andconditions mediated or associated with PCSK9 activity. This particularlyprovides for tailored therapies, prophylaxis and diagnosis in humansthat are devoid of the common PCSK9 gene or protein (ie, the form a ora′ as used in US20120093818A1 and US20110065902A1 to generateantibodies).

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer PCSK9 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), in many of the examples determined by the inventor,comprise multiple changes at the nucleotide and amino acid levels fromthe corresponding common form nucleotide and amino acids sequences, ie,there are multiple non-synonymous changes at the nucleotide level thattranslate into multiple corresponding changes in the protein target inhumans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor realised that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-PCSK9 ligand for administration to human patients fortherapy and/or prophylaxis of PCSK9-mediated or associated diseases orconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, the present inventor decided to determine aset of human PCSK9 variants on the basis of the following criteria,these being criteria that the inventor realised would provide for usefulmedical drugs and diagnostics to tailored need in the human population.The inventor selected variants having at least 3 of the 4 followingcriteria:—

-   -   PCSK9 variants having a cumulative human allele frequency in the        range from 1 to 10%;    -   PCSK9 variants having a total human genotype frequency in the        range from 1 to about 15%;    -   PCSK9 variants found in many different human ethnic populations        (using the standard categorisation of the 1000 Genomes Project,        which is an accepted standard in the art; see Table 4 below);        and    -   PCSK9 variants found in many individuals distributed across such        many different ethnic populations.

On the basis of these criteria, the inventor identified the variantslisted in Table 1 below (excluding form a).

The inventor's selection included, as a consideration, selection fornucleotide variation that produced amino acid variation in correspondingPCSK9 forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

TABLE 1 Human PCSK9 variants distributed over several human ethnicpopulations & having a total human genotype frequency in the range of 1to about 15% (a) Amino acid variability, population distributions andfrequencies Form a 46R 53A 425N 443A 474I 619Q 670E ASW, YRI, GBR 939 140.3951 0.4506 0.64815 TSI, CLM, CHB (0.8457) LWK, CHS, MXL, JPT, PUR,IBS, FIN, CEU No. Hom Freq⁴ Variant Amino Acid Position & VariationHuman No. Unique (Het + Hom Form 46L 53V 425S 443T 474V 619P 670GPopulations Individs¹ Pops² Het Freq³ freq⁵) Cum Freq⁶ f x ASW, YRI,GBR, 180 12 0.153 0.009 0.0855 TSI, CLM, LWK, (0.162) MXL, JPT, PUR,IBS, FIN, CEU c x ASW, YRI, GBR 153 12 0.1296 0.0081 0.0729 TSI, CLM,CHB, (0.1377) LWK, CHS, JPT, PUR, FIN, CEU r x x 0.0234 0.009 0.0292(0.0324) p x x ASW, GBR, TSI, 49 9 0.0441 (0.0441) 0.0221 CLM, JPT, PUR,IBS, FIN, CEU m x LWK, ASW, YRI 29 4 0.0225 (0.0225) 0.0149 CLM e x xLWK, ASW, YRI 15 3 0.0135 (0.0135) 0.0068 h x x LWK, ASW, YRI 10 3 0.009(0.009) 0.0045 aj X x PUR, TSI, FIN, CEU 9 4 0.0081 (0.0081) 0.0041 q xx CHS, ASW, JPT, 7 5 0.0063 (0.0063) 0.0032 PUR, CHB Table Footnotes:“x” in a box indicates that the amino acid for the variant form isdifferent from the amino acid at that position in form a, the variantamino acid being shown in “Amino Acid Position & Variation” of the tableand the form a amino acid being shown in the first row of the table;amino acids at all other positions of each variant form are identical tothose found in form a. Amino acid numbering is per the numbering shownfor the pro-form in Table 2 below. ¹Number of individuals in 1000Genomes database found to have the allele; ²Number of unique humanethnic populations in 1000 Genomes database in which the allele wasfound to occur; ³Heterozygous human genotype frequency, ie, cumulativefrequency of all genotypes having one occurrence of the variant alleleand one occurrence of another allele (heterozygous state), eg, acgenotype in 1000 Genomes database; ⁴Homozygous human genotype frequency,ie, cumulative frequency of two occurrences of the variant allele(homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Totalhuman genotype frequency, ie, total of heterozygous plus homozygoushuman genotype frequencies. ⁶Cumulative human allele frequency of alloccurrences of the variant allele in 1000 Genomes database. Form a′ isidentical to form a with the exception that form a' has a glycine (G) atposition 620 (see US20120093818 (Amgen, Inc)); form a has E at thisposition . (b) Nucleotide Sequence Variations of Selected Alleles Allelea G C A G A A A Nucleotide Position¹ Variant 1:55505647 1:555056681:55523802 1:55523855 1:55524237 1:55527222 1:55529187 AlleleNon-Synonymous Nucleotide Variation² T T G A G C G Variant ID³rs11591147 rs11583680 rs28362261 rs28362263 rs562556 rs28362277 rs505151Corresponding Amino Acid Variation 46L 53V 425S 443T 474V 619P 670G f Xc X r X X p X X m X e X X h X X aj X X q X X “x” in a box indicates thata variant allele comprises the non-synonymous nucleotide variationindicated in the 5th row. Table Footnotes: ¹Notation is chromosomenumber (all positions are on human chromosome 1):coordinate number(Ensembl release 73 - September 2013, Genome assembly: GRCh37(GCA_000001405.13); ²Nucleotide change (compared to allele a nucleotideshown in first row) giving rise to an amino acid change in the variantform (compared to amino acid of allele a); and ³NCBI dbSNP referencenumber (NCBI dbSNP Build 138 released on Apr 25, 2013).

TABLE 2 Sequences (a) Human PCSK9 Form a Amino Acid Sequence (SEQ IDNO: 1) - “Pro-form” with Signal Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtlgtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAESMGTRVHCHQQGHVLTGCSSHWEVEDLGT

Italics = signal sequence 1-30

lower case = catalytic domain 153-449 UPPER CASE = C-terminal domain450-692 Underlined = residues changed from allele a in other sequences(aa residue number shown) The pro-form is the sequence from amino acidnumber 31 to (and including) amino acid number 692 of SEQ ID NO: 1. Themature form is the sequence from amino acid number 153 to (andincluding) amino acid number 692 of SEQ ID NO: 12 (b) Human PCSK9 Form aAmino Acid Sequence (SEQ ID NO: 3) - “Mature-form” (Numbering andnotation as per SEQ ID NO: 1 above has been retained)sipwnleritppryradeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgthlagvvsgrdagvakgasmrslrvlncqgkgtvsgtliglefirksqlvqpvgplvvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtlgtltnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammlsaepeltlaelrqrlihfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

(c) Human PCSK9 Allele a Nucleotide Sequence (SEQ ID NO: 28) - Encoding“Pro-form” Plus Signal SequenceATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtcgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

GCCTCCCAGGAGCTCCAGTGAC Italics = nucleotide sequence encoding signalsequence (nucleotides 1-90)

lower case = nucleotide sequence encoding catalytic domain (nucleotides457-1346) UPPER CASE = nucleotide sequence encoding C-terminal domain(nucleotides 1347-2076) Underlined = allelic variations from allele a inother sequences (aa residue number changes and codon changes shown) Thepro-form is encoded by nucleotide sequence from nucleotide 91 to (andincluding) nucleotide 2076. The mature form is encoded by nucleotidesequence from nucleotide 457 to (and including) nucleotide 2076.

Variant Allele Nucleotide Sequences

Thus,

(i) The nucleotide sequence of allele f is identical to SEQ ID NO: 28except that the nucleotide sequence of allele f comprises a GTC codoninstead of an ATC codon at the position labelled “I474V” in SEQ ID NO:28;

-   (ii) The nucleotide sequence of allele c is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele c comprises a GGG    codon instead of an GAG codon at the position labelled “E670G” in    SEQ ID NO: 28;-   (iii) The nucleotide sequence of allele r is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele r comprises a GTC    codon instead of an ATC codon at the position labelled “I474V” in    SEQ ID NO: 28; and a GGG codon instead of an GAG codon at the    position labelled “E670G” in SEQ ID NO: 28;-   (iv) The nucleotide sequence of allele p is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele p comprises a GTC    codon instead of a GCC codon at the position labelled “A53V” in SEQ    ID NO: 28; and a GTC codon instead of an ATC codon at the position    labelled “I474V” in SEQ ID NO: 28;-   (v) The nucleotide sequence of allele m is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele m comprises a ACC    codon instead of a GCC codon at the position labelled “A443T” in SEQ    ID NO: 28;-   (vi) The nucleotide sequence of allele e is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele e comprises a AGT    codon instead of an AAT codon at the position labelled “N425S” in    SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the    position labelled “I474V” in SEQ ID NO: 28;-   (vii) The nucleotide sequence of allele h is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele h comprises a ACC    codon instead of a GCC codon at the position labelled “A443T” in SEQ    ID NO: 28; and a CCG codon instead of a CAG codon at the position    labelled “Q619P” in SEQ ID NO: 28;-   (viii) The nucleotide sequence of allele aj is identical to SEQ ID    NO: 28 except that the nucleotide sequence of allele aj comprises a    CTT codon instead of an CGT codon at the position labelled “R46L” in    SEQ ID NO: 28; and a GTC codon instead of an ATC codon at the    position labelled “I474V” in SEQ ID NO: 28; and-   (ix) The nucleotide sequence of allele q is identical to SEQ ID NO:    28 except that the nucleotide sequence of allele q comprises a GTC    codon instead of a GCC codon at the position labelled “A53V” in SEQ    ID NO: 28; and a GGG codon instead of an GAG codon at the position    labelled “E670G” in SEQ ID NO: 28.

Variant Pro-Form Amino Acid Sequences

(Numbering is as Per SEQ ID NO: 1 Recited Above)

(A) The amino acid sequence of form f is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form fcomprises a valine at position 474;

(B) The amino acid sequence of form c is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ccomprises a glycine at position 670;

(C) The amino acid sequence of form r is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form rcomprises a valine at position 474 and a glycine at position 670;(D) The amino acid sequence of form p is identical the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form pcomprises a valine at position 53 and a valine at position 474;(E) The amino acid sequence of form m is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form mcomprises a threonine at position 443;(F) The amino acid sequence of form e is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ecomprises a serine at position 425 and a valine at position 474;(G) The amino acid sequence of form h is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form hcomprises a threonine at position 443 and a proline at position 619;(H) The amino acid sequence of form aj is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form ajcomprises a leucine at position 46 and a valine at position 474; and(I) The amino acid sequence of form q is identical to the amino acidsequence from amino acid number 31 to (and including) amino acid number692 of SEQ ID NO: 1 except that the amino acid sequence of form qcomprises a valine at position 53 and a glycine at position 670.

Variant Mature Form Amino Acid Sequences

(Numbering is as per SEQ ID NO: 1 recited above)

(A′) The amino acid sequence of form f is identical to SEQ ID NO: 2except that the amino acid sequence of form f comprises a valine atposition 474;

(B′) The amino acid sequence of form c is identical to SEQ ID NO: 2except that the amino acid sequence of form c comprises a glycine atposition 670;

(C′) The amino acid sequence of form r is identical to SEQ ID NO: 2except that the amino acid sequence of form r comprises a valine atposition 474 and a glycine at position 670;

(D′) The amino acid sequence of form p is identical to SEQ ID NO: 2except that the amino acid sequence of form p comprises a valine atposition 474;

(E′) The amino acid sequence of form m is identical to SEQ ID NO: 2except that the amino acid sequence of form m comprises a threonine atposition 443;

(F′) The amino acid sequence of form e is identical to SEQ ID NO: 2except that the amino acid sequence of form e comprises a serine atposition 425 and a valine at position 474;

(G′) The amino acid sequence of form h is identical to SEQ ID NO: 2except that the amino acid sequence of form h comprises a threonine atposition 443 and a proline at position 619;

(H′) The amino acid sequence of form aj is identical to SEQ ID NO: 2except that the amino acid sequence of form aj comprises valine atposition 474; and

(I′) The amino acid sequence of form q is identical to SEQ ID NO: 2except that the amino acid sequence of form q comprises a glycine atposition 670.

The mature form of p is identical to the mature form off and aj.

The mature form of c is identical to the mature form of q.

Further sequence analysis and 3D in silico modelling (see FIG. 1)revealed that selected variants also fulfilled the following selectioncriteria:—

-   -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are found in the mature form of the        target (ie, outside the pro-domain); and    -   PCSK9 variants whose variant amino acid residues (versus the        common form of human PCSK9) are surface-exposed on the target,        which the inventor saw as contributing to determining the        topography of the target and potentially contributing to how and        where ligand binding on the target occurs.

As shown in FIG. 1, identified positions 425, 443, 474, 619 and 670(found in the selected variants of the invention) are allsurface-exposed and outside of the pro-domain. Variant positions 425 and443 are surface-exposed on the catalytic domain, while variant positions474, 619 and 670 are surface-exposed on the C-terminal domain.

In a first example, the invention addresses the need to treat humanshaving naturally-occurring rarer natural PCSK9 alleles, genotypes andphenotypes (rarer protein forms). In this respect, the inventionprovides the following aspects:

In a First Aspect: An anti-human PCSK9 ligand for use in a method oftreating and/or preventing a PCSK9-mediated disease or condition in ahuman whose genome comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37, wherein the method comprisesadministering the ligand to the human.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In the alternative the nucleotide sequence encodes a human PCSK9comprising a mutation R46L, A53V, N425S, A443T, I474V, Q619P and E670G(eg, comprises a mutation I474, E670G, N425S or Q619P) in SEQ ID NO: 1;for example, the PCSK9 comprises I474V in SEQ ID NO: 1 and optionallythe human comprises such a PCSK9 or a nucleotide sequence encoding sucha PCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg reducing cholesterol or maintaining reduced cholesterolin the human); for example, the PCSK9 comprises E670G in SEQ ID NO: 1and optionally the human comprises such a PCSK9 or a nucleotide sequenceencoding such a PCSK9 (eg, wherein the method is for treating orpreventing dislipidemia, eg reducing cholesterol or maintaining reducedcholesterol in the human); for example, the PCSK9 comprises Q619P in SEQID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotidesequence encoding such a PCSK9 (eg, wherein the method is for treatingor preventing dislipidemia, eg reducing cholesterol or maintainingreduced cholesterol in the human); for example, the PCSK9 comprisesN425S in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 ora nucleotide sequence encoding such a PCSK9 (eg, wherein the method isfor treating or preventing dislipidemia, eg reducing cholesterol ormaintaining reduced cholesterol in the human); for example, the PCSK9comprises R46L in SEQ ID NO: 1 and optionally the human comprises such aPCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein themethod is for treating or preventing dislipidemia, eg increaingcholesterol in the human); for example, the PCSK9 comprises A53V in SEQID NO: 1 and optionally the human comprises such a PCSK9 or a nucleotidesequence encoding such a PCSK9 (eg, wherein the method is for treatingor preventing dislipidemia, eg increaing cholesterol in the human); forexample, the PCSK9 comprises A443T in SEQ ID NO: 1 and optionally thehuman comprises such a PCSK9 or a nucleotide sequence encoding such aPCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg increaing cholesterol in the human).

In a Second Aspect: The ligand of aspect 1, wherein the ligand has beenor is determined as capable of binding a human PCSK9 selected from thegroup consisting forms f c, r, p, m, e, h, aj and q.

In the alternative the ligand has been or is determined as capable ofbinding a human PCSK9 comprising a mutation R46L, A53V, N425S, A443T,I474V, Q619P and E670G (eg, comprises a mutation I474, E670G, N425S orQ619P) in SEQ ID NO: 1; for example, the PCSK9 comprises I474V in SEQ IDNO: 1 and optionally the human comprises such a PCSK9 or a nucleotidesequence encoding such a PCSK9 (eg, wherein the method is for treatingor preventing dislipidemia, eg reducing cholesterol or maintainingreduced cholesterol in the human); for example, the PCSK9 comprisesE670G in SEQ ID NO: 1 and optionally the human comprises such a PCSK9 ora nucleotide sequence encoding such a PCSK9 (eg, wherein the method isfor treating or preventing dislipidemia, eg reducing cholesterol ormaintaining reduced cholesterol in the human); for example, the PCSK9comprises Q619P in SEQ ID NO: 1 and optionally the human comprises sucha PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, wherein themethod is for treating or preventing dislipidemia, eg reducingcholesterol or maintaining reduced cholesterol in the human); forexample, the PCSK9 comprises N425S in SEQ ID NO: 1 and optionally thehuman comprises such a PCSK9 or a nucleotide sequence encoding such aPCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg reducing cholesterol or maintaining reduced cholesterolin the human); for example, the PCSK9 comprises R46L in SEQ ID NO: 1 andoptionally the human comprises such a PCSK9 or a nucleotide sequenceencoding such a PCSK9 (eg, wherein the method is for treating orpreventing dislipidemia, eg increaing cholesterol in the human); forexample, the PCSK9 comprises A53V in SEQ ID NO: 1 and optionally thehuman comprises such a PCSK9 or a nucleotide sequence encoding such aPCSK9 (eg, wherein the method is for treating or preventingdislipidemia, eg increaing cholesterol in the human); for example, thePCSK9 comprises A443T in SEQ ID NO: 1 and optionally the human comprisessuch a PCSK9 or a nucleotide sequence encoding such a PCSK9 (eg, whereinthe method is for treating or preventing dislipidemia, eg increaingcholesterol in the human).

In an example of any aspect, the ligand binds (or has been determined tobind) two, three, four or more human PCSK9 selected from the groupconsisting forms f c, r, p, m, e, h, aj and q or said PCSK9s in saidalternative.

In an example of any aspect, the ligand comprises a protein domain thatspecifically binds to PCSK9, eg, a human PCSK9 selected from the groupconsisting forms f c, r, p, m, e, h, aj and q, or said PCSK9s in saidalternative.

The term “specifically binds,” or the like, means that a ligand, eg, anantibody or antigen-binding fragment thereof, forms a complex with anantigen that is relatively stable under physiologic conditions. Specificbinding can be characterized by an equilibrium dissociation constant ofat least about 1×10⁻⁶M or less (e.g., a smaller KD denotes a tighterbinding). Methods for determining whether two molecules specificallybind are well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds a human PCSK9 may, however, exhibitcross-reactivity to other antigens such as a PCSK9 molecule from anotherspecies. Moreover, multi-specific antibodies (e.g., bispecifics) thatbind to human PCSK9 and one or more additional antigens are nonethelessconsidered antibodies that “specifically bind” PCSK9, as used herein.

In an example of any aspect, the ligand comprises or consists of aprotein that mimics the EGFA domain of the LDL receptor and specificallybinds to PCSK9, eg, a human PCSK9 selected from the group consistingforms f c, r, p, m, e, h, aj and q, or said PCSK9s in said alternative.

In an example of any aspect, the ligand antagonises PCSK9, eg, a humanPCSK9 selected from the group consisting forms f c, r, p, m, e, h, ajand q, or said PCSK9s in said alternative.

In an example of any aspect, the method comprises (before administeringthe ligand) the step of determining that the ligand is capable ofbinding a human PCSK9 selected from the group consisting forms f c, r,p, m, e, h, aj and q, or said PCSK9s in said alternative.

In an example of any aspect, binding is determined by SPR. In an exampleof any aspect, binding is determined by ELISA.

In an example of any aspect, said forms are the mature forms.

In an example of any aspect, said forms are the pro-forms.

In a Third Aspect: A ligand that binds (i) a PCSK9 of said alternativeor (ii) a human PCSK9 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4-27 for use in a method comprisingthe step of using the ligand to target said PCSK9 in a human to treatand/or prevent a disease or condition mediated by PCSK9, the methodcomprising administering the ligand to the human.

In an example, the disease or condition is mediated by (i) a PCSK9 ofsaid alternative or (ii) a human PCSK9 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C(Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above.

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Fourth Aspect: The ligand of aspect 3, wherein the genome of thehuman comprises a nucleotide sequence selected from the group consistingof SEQ ID NOs: 29-37; or a nucleotide sequence encoding a PCSK9 of saidalternative.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33

In an example, the nucleotide sequence is SEQ ID NO: 34

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Fifth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is genotyped as positive for a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Sixth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is phenotyped as positive for (i) a PCSK9 of saidalternative or (ii) a human PCSK9 selected from the group consisting offorms f c, r, p, m, e, h, aj and q or at least the catalytic orC-terminal domain thereof.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Seventh Aspect: The ligand of any preceding aspect, wherein themethod comprises genotyping the human as positive for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or atleast the catalytic domain- or C-terminal domain-encoding sequencethereof; or a nucleotide sequence encoding a PCSK9 of said alternative.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eighth Aspect: The ligand of any preceding aspect, wherein themethod comprises phenotyping the human has positive for (i) a PCSK9 ofsaid alternative or (ii) a human PCSK9 selected from the groupconsisting of forms f c, r, p, m, e, h, aj and q or at least thecatalytic or C-terminal domain thereof

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Ninth Aspect: The ligand of any preceding aspect, wherein the humanhas been or is genotyped as heterozygous for a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof, or anucleotide sequence encoding a PCSK9 of said alternative; optionallywherein the human has been or is genotyped as comprising the nucleotidesequence of SEQ ID NO: 28 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof and a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof; or anucleotide sequence encoding a PCSK9 of said alternative.

“Heterozygous” here means that in the human's genotype one allelecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof and other allele can be any PCSK9 (eg,form a, a′ or an allele comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof).

In an example, the method comprises (before administering the ligand)genotyping the human as heterozygous for a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 29-37 or at least the catalyticdomain- or C-terminal domain-encoding sequence thereof; optionally alsogenotyping the human as comprising the nucleotide sequence of SEQ ID NO:28 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof and a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37.

These are naturally-occurring allele (haplotype) sequences that do notencode 46L and which meet the criteria set out above. These groupscomprise variants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Tenth Aspect: The ligand of any one of aspects 1 to 9, wherein thegenome of the human has been or is genotyped as homozygous for anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or at least the catalytic domain- or C-terminal domain-encodingsequence thereof; or a nucleotide sequence encoding a PCSK9 of saidalternative.

“Homozygous” here means that in the human's genotype each allelecomprises the same nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof.

In an example, the method comprises genotyping the human as homozygousfor a nucleotide sequence selected from the group consisting of SEQ IDNOs: 29-37 or at least the catalytic domain- or C-terminaldomain-encoding sequence thereof; or a nucleotide sequence encoding aPCSK9 of said alternative.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an Eleventh Aspect: The ligand of any preceding aspect, wherein theligand comprises an antibody binding site that binds (i) a PCSK9 of saidalternative or (ii) a human PCSK9 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4-27 and optionallyhas been or is determined as capable of such binding.

In an example, the method comprises (before administering the ligand)the step of determining that the ligand is capable of binding to saidhuman PCSK9.

In an example, the binding is specific binding. In an example, theligand binds (or has been determined as binding) to the PCSK9 with anaffinity (Kd) of 1 mM, 100 nM, lOnM or 1 nM or less. In an embodiment,the affinity is no less than 10, 100 or 1000 fM.

In an example, binding or affinity is determined by SPR or ELISA.

In an example, the disease or condition is mediated by a human PCSK9comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4-27.

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4-23, 26 and 27; or selected from the groupconsisting of SEQ ID NOs: 4-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 4-14, 18-23, 26 and 27. These arenaturally-occurring sequences that do not comprise 46L and which meetthe criteria set out above. These groups comprise variants that areassociated with elevated LDL-C.

In an example, the amino acid sequence is SEQ ID NO: 18, 19 or 20, thatcomprises a 425S, which is associated with elevated LDL-C(Pisciotta etal 2006).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10, 11, 12, 26 and 27, that comprise 670Gwhich is a marker for severity of coronary atherosclerosis (Chen et al2005).

In an example, the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-14 and 18-27; or selected from the groupconsisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequencesthat have a naturally-occurring combination of differences from SEQ IDNOs: 1-3 (form a) and which meet the criteria set out above

In an example, the amino acid sequence is SEQ ID NO: 4.

In an example, the amino acid sequence is SEQ ID NO: 5.

In an example, the amino acid sequence is SEQ ID NO: 6.

In an example, the amino acid sequence is SEQ ID NO: 7.

In an example, the amino acid sequence is SEQ ID NO: 8.

In an example, the amino acid sequence is SEQ ID NO: 9.

In an example, the amino acid sequence is SEQ ID NO: 10.

In an example, the amino acid sequence is SEQ ID NO: 11.

In an example, the amino acid sequence is SEQ ID NO: 12.

In an example, the amino acid sequence is SEQ ID NO: 13.

In an example, the amino acid sequence is SEQ ID NO: 14.

In an example, the amino acid sequence is SEQ ID NO: 15.

In an example, the amino acid sequence is SEQ ID NO: 16.

In an example, the amino acid sequence is SEQ ID NO: 17.

In an example, the amino acid sequence is SEQ ID NO: 18.

In an example, the amino acid sequence is SEQ ID NO: 19.

In an example, the amino acid sequence is SEQ ID NO: 20.

In an example, the amino acid sequence is SEQ ID NO: 21.

In an example, the amino acid sequence is SEQ ID NO: 22.

In an example, the amino acid sequence is SEQ ID NO: 23.

In an example, the amino acid sequence is SEQ ID NO: 24.

In an example, the amino acid sequence is SEQ ID NO: 25.

In an example, the amino acid sequence is SEQ ID NO: 26.

In an example, the amino acid sequence is SEQ ID NO: 27.

In a Twelfth Aspect: The ligand of aspect 11, wherein the ligand is anantibody or antibody fragment. For example, the antibody or antibodyfragment is a PCSK9 antagonist, eg, neutralises PCSK9.

Examples of such antibodies are disclosed, for instance, in WO2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO2008/133647, WO 2009/100297, WO 2009/100318, WO 2011/037791, WO2011/053759, WO 2011/053783, WO 2008/125623, WO 2011/072263, WO2009/055783, WO 2010/029513, WO 2011/11 1007, WO 2010/077854, thedisclosures and sequences of such antibodies being incorporated hereinfor use in the invention in their entireties by reference. One specificexample is AMG 145 (Amgen), LY3015014 (Eli Lilly) or alirocumab.Advantageously, the ligand is or comprises alirocumab. Alternatively,the ligand is or comprises evolocumab.

In an example, the ligand is SAR236553/REGN727 (SanofiAventis/Regeneron) or a PCSK9-binding derivative thereof.

In an example, the ligand comprises or consists of a neutralizingantibody that binds to the PCSK9, wherein the antibody binds to PCSK9and reduces the likelihood that PCSK9 binds to LDLR.

The ligand of aspect 11, wherein the ligand is a PCSK9 antagonist, eg,neutralises PCSK9.

In an example of any aspect of the invention, the ligand comprises orconsists a ligand selected from evolocumab, 1D05-IgG2 (Merck & Co.),ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat), LY3015014 (Eli Lilly) andalirocumab (SAR236553/REGN727; Sanofi Aventis/Regeneron).

In Thirteenth Aspect: The ligand of any one of aspects 1 to 10, wherein(i) the ligand comprises a sequence of contiguous nucleotides thatspecifically hybridises to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or at least the catalytic domain- orC-terminal domain-encoding sequence thereof (or hybridizes to anucleotide sequence encoding a PCSK9 of said alternative), orspecifically hybridises to an antisense sequence or an RNA transcript ofsaid sequence, wherein said sequence of contiguous nucleotideshybridises to at least one nucleotide present in said selected sequencewhich is not present in SEQ ID NO: 28 or hybridises to an antisensesequence or an RNA transcript thereof respectively; and/or (ii) theligand comprises a sequence of at least 10 contiguous nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 thereof (or a nucleotide sequence encoding a PCSK9 of saidalternative) or is an antisense sequence or RNA version of saidcontiguous nucleotides, wherein said sequence of contiguous nucleotidescomprises at least one nucleotide present in said selected sequencewhich is not present in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In an embodiment, the ligand comprises at least 10, 11, 12, 13, 14, 15,20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of saidnucleotide sequence.

In a Fourteenth Aspect: The ligand of any preceding aspect, wherein saiddisease or condition is hyperlipidaemia, hypercholesterolaemia (eg,familial hypercholesterolaemia), heart attack, stroke, coronary heartdisease, atherosclerosis or a cardiovascular disease or condition.

The ligand of any preceding aspect, wherein the disease or condition ishypercholesterolemia, hyperlipidemia, hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In an example, said disease or condition is hypercholesterolaemia. Theterm “hypercholesterolaemia,” as used herein, refers to a condition inwhich cholesterol levels are elevated above a desired level. In someembodiments, this denotes that serum cholesterol levels are elevated. Insome embodiments, the desired level takes into account various “riskfactors” that are known to one of skill in the art (and are described orreferenced in US20120093818).

The ligand of any preceding aspect, wherein the human is identified asheterozygous for Familial Hypercholesterolemia, statin intolerant,statin uncontrolled, or at risk for developing hypercholesterolemia,dyslipidemia, cholestatic liver disease, nephrotic syndrome,hypothyroidism, obesity, atherosclerosis or a cardiovascular disease.

In a Fifteenth Aspect: The ligand of any preceding aspect, wherein saiddisease or condition is associated with elevated LDL cholesterol.

Cholesterol levels are measured in milligrams (mg) of cholesterol perdeciliter (dL) of blood in the United States and some other countries.Canada and most European countries measure cholesterol in millimoles(mmol) per liter (L) of blood. Below are general guideline ideal rangesand elevated ranges.

Total cholesterol Total cholesterol* (U.S. and (Canada some othercountries) and most of Europe) Below 200 mg/dL Below 5.2 mmol/L Ideal200-239 mg/dL 5.2-6.2 mmol/L Borderline high 240 mg/dL and above Above6.2 mmol/L High LDL cholesterol LDL cholesterol* (U.S. and (Canada someother countries) and most of Europe) 100-129 mg/dL 2.6-3.3 mmol/L Ideal130-159 mg/dL 3.4-4.1 mmol/L Borderline high 160-189 mg/dL 4.1-4.9mmol/L High 190 mg/dL and above Above 4.9 mmol/L Very high *Canadian andEuropean guidelines differ slightly from U.S. guidelines. Theseconversions are based on U.S. guidelines.

Elevated LDL cholesterol is, therefore, 160 mg/dL or above (4.1 mmol/Lor above).

In a Sixteenth Aspect: The ligand of any preceding aspect, wherein theligand inhibits human PCSK9 binding to human LDL receptor and optionallyhas been or is determined as capable of such inhibition.

In an example, the method comprises (before administering the ligand)determining that the ligand is capable of such inhibition.

Inhibition determination is eg, inhibition in a blood or serum sample,at rtp, at ph7, at 37 degrees centigrade and/or under the physiologicalconditions of a human body.

In a Seventeeth Aspect: The ligand of any preceding aspect, wherein thehuman is resistant or substantially resistant to statin (eg, avorstatinand/or fluvastatin) treatment of said disease or condition.

In an Eighteenth Aspect: The ligand of any preceding aspect, wherein theligand is for treating and/or preventing a PCSK9-mediated disease orcondition in a human

(i) whose genome comprises SEQ ID NO: 29 and wherein the human is ofASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry;or

(ii) whose genome comprises SEQ ID NO: 30 and wherein the human is ofASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry;or

(iii) whose genome comprises SEQ ID NO: 32 and wherein the human is ofASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or

(iv) whose genome comprises SEQ ID NO: 33 and wherein the human is ofLWK, ASW, YRI or CLM ancestry; or

(v) whose genome comprises SEQ ID NO: 34 and wherein the human is ofLWK, ASW or YRI ancestry; or

(vi) whose genome comprises SEQ ID NO: 35 and wherein the human is ofPUR, TSI, FIN or CEU ancestry; or

(vii) whose genome comprises SEQ ID NO: 36 and wherein the human is ofLWK, ASW or YRI ancestry; or

(viii) whose genome comprises SEQ ID NO: 37 and wherein the human is ofCHS, ASW, JPT, PUR or CHB ancestry.

In a Ninteenth Aspect: The ligand of any preceding aspect, wherein theligand is for treating and/or preventing a PCSK9-mediated disease orcondition in a human

(i) that expresses PCSK9 form f and wherein the human is of ASW, YRI,GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU ancestry; or

(ii) that expresses PCSK9 form c and wherein the human is of ASW, YRI,GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU ancestry; or

(iii) that expresses PCSK9 form p and wherein the human is of ASW, GBR,TSI, CLM, JPT, PUR, IBS, FIN or CEU ancestry; or

(iv) that expresses PCSK9 form m and wherein the human is of LWK, ASW,YRI or CLM ancestry; or

(v) that expresses PCSK9 form e and wherein the human is of LWK, ASW orYRI ancestry; or

(vi) that expresses PCSK9 form h and wherein the human is of PUR, TSI,FIN or CEU ancestry; or

(vii) that expresses PCSK9 form aj and wherein the human is of LWK, ASWor YRI ancestry; or

(viii) that expresses PCSK9 form q and wherein the human is of CHS, ASW,JPT, PUR or CHB ancestry.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twentieth Aspect: A pharmaceutical composition or kit for treatingand/or preventing a PCSK9-mediated condition or disease (eg, as recitedin aspect 14 or 15), the composition or kit comprising a ligand of anypreceding aspect and optionally a statin (eg, cerovastatin,atorvastatin, simvastatin, pitavastin, rosuvastatin, fluvastatin,lovastatin or pravastatin); and optionally in combination with a labelor instructions for use to treat and/or prevent said disease orcondition in a human (eg, covering treatment of a human as recited inaspect 18 or 19); optionally wherein the label or instructions comprisea marketing authorisation number (eg, an FDA or EMA authorisationnumber); optionally wherein the label or instructions comprisedirections to administer alirocumab or evolocumab to said human;optionally wherein the kit comprises an IV or injection device thatcomprises the ligand (and, eg, also a statin).

In a Twenty-first Aspect: A method of producing an anti-human PCSK9antibody binding site, the method comprising obtaining a plurality ofanti-PCSK9 antibody binding sites, screening the antibody binding sitesfor binding to (i) a PCSK9 of said alternative or (ii) a human PCSK9selected from the group consisting of forms f c, r, p, m, e, h, aj and qor a catalytic or C-terminal domain or a peptide thereof that comprisesamino acid variation from the corresponding sequence of SEQ ID NO: 1, 2or 3 and isolating an antibody binding site that binds in the screeningstep, and optionally producing a form f c, r, p, m, e, h, aj or qPCSK9-binding fragment or derivative of the isolated antibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example of this and the next aspect, the plurality of bindingsites comprises or consists of a plurality of 4-chain antibodies orfragments thereof, eg, dAbs, Fabs or scFvs. Suitable methods forproducing pluralities of binding sites for screening include phagedisplay (producing a phage display library of antibody binding sites),ribosome display (producing a ribosome display library of antibodybinding sites), yeast display (producing a yeast display library ofantibody binding sites), or immunisation of a non-human vertebrate (eg,a rodent, eg, a mouse or rat, eg, a Velocimouse™, Kymouse™, Xenomouse™,Aliva Mouse™, HuMab Mouse™, Omnimouse™, Omnirat™ or MeMo Mouse™) with aPCSK9 epitope and isolation of a repertoire of antibody-producing cells(eg, a B-cell, plasma cell or plasmablast repertoire) and/or arepertoire of isolated antibodies.

In an example, the method comprises selecting one or more antibodybinding sites that each specifically binds to a human PCSK9 epitopecomprising amino acid variation from the corresponding sequence of SEQID NO: 1, 2 or 3.

In a Twenty-second Aspect: A method of producing an anti-human PCSK9antibody, the method comprising immunising a non-human vertebrate (eg, amouse or a rat) with (i) a PCSK9 of said alternative or (ii) a humanPCSK9 comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of forms f c, r, p, m, e, h, ajand q or a catalytic or C-terminal domain or a peptide thereof thatcomprises amino acid variation from the corresponding sequence of SEQ IDNO: 1, 2 or 3 and isolating an antibody that binds a human PCSK9comprising selected from the group consisting of forms f c, r, p, m, e,h, aj and q or a catalytic or C-terminal domain or a peptide thereofthat comprises amino acid variation from the corresponding sequence ofSEQ ID NO: 1, 2 or 3, and optionally producing a form f c, r, p, m, e,h, aj or q PCSK9-binding fragment or derivative of the isolatedantibody.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-third Aspect: The method of aspect 21 or 22, comprising thestep of obtaining a nucleic acid encoding the antibody, fragment,derivative or binding site and optionally inserting the nucleic acid inan expression vector.

For example, the method comprises isolating a cell (eg, B-cell,plasmablast, plasma cell or memory cell) comprising the nucleic acid,wherein the cell is obtained from a non-human vertebrate that has beenimmunised with the PCSK9 epitope.

In a Twenty-fourth Aspect: A kit for PCSK9 genotyping a human, whereinthe kit comprises a nucleic acid (i) comprising a sequence of 10 or more(eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) contiguousnucleotides that specifically hybridises to (i) a nucleotide sequenceencoding a PCSK9 of said alternative or (ii) a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 29-37 or at least thecatalytic domain- or C-terminal domain-encoding sequence thereof, orspecifically hybridises to an antisense sequence or an RNA transcript ofsaid sequence, wherein said sequence of contiguous nucleotideshybridises to at least one nucleotide present in said selected sequencewhich is not present in SEQ ID NO: 28 or hybridises to an antisensesequence or an RNA transcript thereof; and/or (ii) comprising a sequenceof at least 10 or more (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100or more) nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 (or the nucleotide sequence of (i)) orcomprising an antisense sequence or RNA version of said contiguousnucleotides, wherein said sequence of contiguous nucleotides comprisesat least one nucleotide present in said selected sequence which is notpresent in SEQ ID NO: 28.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Twenty-fifth Aspect: A kit for PCSK9 genotyping or phenotyping ahuman, wherein the kit comprises a ligand according to any one ofaspects 1 to 19 or an antibody, fragment or derivative produced by themethod of any one of aspects 21 to 23.

In a Twenty-sixth Aspect: Use of an anti-PCSK9 ligand that binds (i) aPCSK9 of said alternative or (ii) a human PCSK9 selected from the groupconsisting of forms f c, r, p, m, e, h, aj and q in the manufacture of amedicament for treating and/or preventing a PCSK9-mediated disease orcondition in a human whose genome comprises (iii) a nucleotide sequenceencoding said PCSK9 of (i); or (iv) a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37, optionally for treatingand/or preventing a PCSK9-mediated disease or condition in a human asrecited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Twenty-seventh Aspect: Use of an anti-PCSK9 ligand that binds (i) aPCSK9 of said alternative or (ii) a human PCSK9 selected from the groupconsisting of forms f, c, r, p, m, e, h, aj and q in the manufacture ofa medicament for targeting said PCSK9 in a human to treat and/or preventa disease or condition mediated by PCSK9, optionally for targeting PCSK9in a human as recited in aspect 18 or 19.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Twenty-eight Aspect: The use of aspect 26 or 27, wherein theligand, human, disease or condition is according to any one of aspects 1to 19.

In a Twenty-ninth Aspect: A method of targeting a PCSK9 for treatingand/or preventing a PCSK9-mediated disease or condition in a human, themethod comprising administering an anti-PCSK9 ligand to a humancomprising (i) a nucleotide sequence encoding a PCSK9 of saidalternative or (ii) a nucleotide sequence selected from the groupconsisting SEQ ID NOs: 29-37, whereby a PCSK9 encoded by said nucleotidesequence is targeted.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirtieth Aspect: The method of aspect 29, wherein the methodcomprises targeting a human PCSK9 selected from the group consisting offorms f c, r, p, m, e, h, aj and q with said ligand to treat and/orprevent said disease or condition in said human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-first Aspect: A method of treating and/or preventing adisease or condition mediated by PCSK9 in a human, the method comprisingtargeting (i) a PCSK9 of said alternative or (ii) a human PCSK9 selectedfrom the group consisting of forms f, c, r, p, m, e, h, aj and q byadministering to the human a ligand that binds said PCSK9 therebytreating and/or preventing said disease or condition in the human.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

The ligand can be any anti-PCSK9 ligand disclosed herein.

In a Thirty-second Aspect: The method of aspect 31, wherein the genomeof the human comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37.

In an example, the nucleotide sequence is selected from the groupconsisting of SEQ ID NOs: 29-35 and 37; or selected from the groupconsisting of SEQ ID NOs: 29-32 and 34-37; or selected from the groupconsisting of SEQ ID NOs: 29-32, 34, 35 and 37. These arenaturally-occurring allele (haplotype) sequences that do not encode 46Land which meet the criteria set out above. These groups comprisevariants that are associated with elevated LDL-C.

In an example, the nucleotide sequence is SEQ ID NO: 34, that encodes a425S, which is associated with elevated LDL-C(Pisciotta et al 2006).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31 and 37, that encode 670G which is a markerfor severity of coronary atherosclerosis (Chen et al 2005).

In an example, the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected fromthe group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These areallele (haplotype) sequences that have a naturally-occurring combinationof differences from SEQ ID NO: 28 (form a) and which meet the criteriaset out above.

In an example, the nucleotide sequence is SEQ ID NO: 29.

In an example, the nucleotide sequence is SEQ ID NO: 30.

In an example, the nucleotide sequence is SEQ ID NO: 31.

In an example, the nucleotide sequence is SEQ ID NO: 32.

In an example, the nucleotide sequence is SEQ ID NO: 33.

In an example, the nucleotide sequence is SEQ ID NO: 34.

In an example, the nucleotide sequence is SEQ ID NO: 35.

In an example, the nucleotide sequence is SEQ ID NO: 36.

In an example, the nucleotide sequence is SEQ ID NO: 37.

In a Thirty-third Aspect: The method of any one of aspects 29 to 32,wherein the human has been or is genotyped as positive for a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or thecatalytic- or C-terminal domain-encoding sequence thereof

In a Thirty-fourth Aspect: The method of any one of aspects 29 to 33,wherein the human has been or is phenotyped as positive for (i) a PCSK9of said alternative or (ii) a human PCSK9 selected from the groupconsisting of forms f c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-fifth Aspect: The method of any one of aspects 29 to 34,wherein the method comprises genotyping the human as positive for (i) anucleotide sequence encoding a PCSK9 of said alternative or (ii) anucleotide sequence selected from the group consisting of SEQ ID NOs:29-37 or the catalytic- or C-terminal domain-encoding sequence thereof.

In a Thirty-sixth Aspect: The method of any one of aspects 29 to 35,wherein the method comprises phenotyping the human as positive for (i) aPCSK9 of said alternative or (ii) a human PCSK9 sequence selected fromthe group consisting of forms f c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Thirty-seventh Aspect: The method of any one of aspects 29 to 36,wherein the human has been or is genotyped as heterozygous for saidnucleotide sequence of (i) or a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof; optionally wherein the human has beenor is genotyped as comprising the nucleotide sequence of SEQ ID NO: 28or the catalytic- or C-terminal domain-encoding sequence thereof andsaid nucleotide sequence of (i) or a nucleotide sequence selected fromthe group consisting of SEQ ID NOs: 29-37 or the catalytic- orC-terminal domain-encoding sequence thereof.

In a Thirty-eighth Aspect: The method of any one of aspects 29 to 37,wherein the genome of the human has been or is genotyped as homozygousfor said nucleotide sequence of (i) or a nucleotide sequence selectedfrom the group consisting of of SEQ ID NOs: 29-37 or the catalytic- orC-terminal domain-encoding sequence thereof.

In a Thirty-ninth Aspect: The method of any one of aspects 29 to 38,wherein the method comprises genotyping the human for said nucleotidesequence of (i) or a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof before administering the ligand to thehuman, wherein the ligand is determined to be capable of binding to aPCSK9 encoded by said selected sequence.

In a Fortieth Aspect: The method of any one of aspects 29 to 39, whereinthe ligand, human, disease or condition is according to any one ofaspects 1 to 19.

In a Forty-first Aspect: A method according to any one of aspects 29 to40 for treating and/or preventing a condition or disease as recited inaspect 14 or 15, the method comprising administering said ligand and astatin (eg, cerovastatin, atorvastatin, simvastatin, pitavastin,rosuvastatin, fluvastatin, lovastatin or pravastatin) to the human.

In a Forty-second Aspect: The method of aspect 41, wherein the ligandand statin are administered separately.

In a Forty-third Aspect: The method of aspect 41, wherein the ligand andstatin are administered simultaneously.

In a Forty-fourth Aspect: The method of any one of aspects 29 to 43,wherein the ligand is administered by subcutaneous injection.

In a Forty-fifth Aspect: A method of PCSK9 genotyping a nucleic acidsample of a human, the method comprising identifying in the sample thepresence of (i) a nucleotide sequence enoding a PCSK9 of saidalternative or (ii) a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 29-37 or the catalytic- or C-terminaldomain-encoding sequence thereof

In a Forty-sixth Aspect: A method of PCSK9 typing a protein sample of ahuman, the method comprising identifying in the sample the presence of(i) a PCSK9 of said alternative or (ii) a human PCSK9 selected from thegroup consisting of forms f c, r, p, m, e, h, aj and q.

In an example, said forms are the mature forms.

In an example, said forms are the pro-forms.

In a Forty-seventh Aspect: The method of aspect 45 or 46, comprisingobtaining a sample of serum, blood, faeces, hair, tissue, cells, urineor saliva from a human, whereby the nucleic acid or protein sample isobtained and used in the step of identifying said sequence.

In a Forty-eighth Aspect: The method of any one of aspects 45 to 47,comprising using a ligand according to any one of aspects 1 to 19 tocarry out said identifying step.

In a Forty-ninth Aspect: A method of treating and/or preventing in ahuman patient a cardiovascular disease or condition, or a disease orcondition that is associated with elevated LDL cholesterol (eg,hypercholesterolaemia), wherein the patient is receiving or haspreviously received statin treatment for said disease or condition, themethod comprising typing the patient using a method of any one ofaspects 45 to 48 and administering a ligand according to one of aspects1 to 19 whereby the human is treated or said disease or condition isprevented; optionally also reducing or stopping statin treatment.

In an example, said reducing or stopping comprises reducing the doseand/or dosing frequency of statin.

In a Fiftieth Aspect: A diagnostic, therapeutic or prophylactic kitcomprising a ligand that is capable of binding to or has been or isdetermined as capable of binding to an amino acid sequence selected fromSEQ ID NOs: 4-27 and instructions for carrying out the method of any oneof aspects 46 to 49 and/or a label or instructions indicating orcovering administration of the ligand to a human as defined in any oneof aspects 1 to 19.

In a Fifty-first Aspect: A diagnostic, therapeutic or prophylactic kitcomprising a nucleic acid probe comprising a nucleotide sequence thatspecifically hybridises to a nucleotide sequence or (i) or a nucleotidesequence selected from the group consisting of SEQ ID NOs: 29-37 or anantisense sequence or RNA transcript thereof and instructions forcarrying out the method of aspect 45, 47 or 48.

In examples of the present invention, the ligand specifically binds tohuman PCSK9, eg, one or more of (i) the PCSK9s of said alternative or(ii) the rare PCSK9 variants disclosed herein (eg, one, two, three, moreor all mature forms f c, r, p, m, e, h, aj and q) and optionally alsothe a and/or a′ form. For example, the ligand specifically binds tomature form f and/or c as well as form a. For example, the ligandspecifically binds to a human PCSK9 comprising a mutation E670G and alsobinds to a human PCSK9 comprising a mutation I474V, Q619P or N425S inSEQ ID NO: 1. For example, the ligand specifically binds to a humanPCSK9 comprising a mutation E670G and also binds to a human PCSK9comprising a mutation I474V in SEQ ID NO: 1. For example, the ligandspecifically binds to a human PCSK9 comprising a mutation E670G and alsobinds to a human PCSK9 comprising a mutation Q619P in SEQ ID NO: 1. Forexample, the ligand specifically binds to a human PCSK9 comprising amutation E670G and also binds to a human PCSK9 comprising a mutationN425S in SEQ ID NO: 1. Determination of such binding can be performed byany antibody binding test as known in the art, eg, by surface plasmonresonance Binding to each such form is, for example, respectively with aKd of at least 1 mM, 100 nM, 1 nM, 100 pM, 10 pM or 1 pM.

In an example, the ligand binds form a and (i) a PCSK9 of saidalternative or (ii) a PCSK9 selected from the group consisting of formsf c, r, p, m, e, h, aj and q, wherein the ligand binding to saidselected form is with a Kd (determined by SPR) that is at least 60, 70,80, 90 or 95% of the Kd for binding to form a. In an embodiment, bothforms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form f, wherein the ligandbinding to form f is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form c, wherein the ligandbinding to form c is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form r, wherein the ligandbinding to form r is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form p, wherein the ligandbinding to formp is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form m, wherein the ligandbinding to form m is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form e, wherein the ligandbinding to form e is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form h, wherein the ligandbinding to form h is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form aj, wherein the ligandbinding to form aj is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In an example, the ligand binds form a and form q, wherein the ligandbinding to form q is with a Kd (determined by SPR) that is at least 60,70, 80, 90 or 95% of the Kd for binding to form a. In an embodiment,both forms are mature forms. In an embodiment, both forms are pro-forms.

In examples of the present invention, the ligand neutralises humanPCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg,one, two, three, more or all mature forms f c, r, p, m, e, h, aj and q)and optionally also the a and/or a′ form. For example, the ligandneutralises mature form f and/or c as well as form a. Determination ofneutralisation can be performed, for example, by any neutralisationassay method disclosed in US20120093818A1 (Amgen, Inc) orUS20110065902A1 (Regeneron Pharmaceuticals, Inc). Ligands of theinvention that bind or target PCSK9 are useful, for example, fortherapeutic and prophylactic applications disclosed in US20120093818A1and US20110065902A1, these specific disclosures being incorporatedherein by reference for use in the present invention and for possibleinclusion in claims herein.

In embodiments where the ligand is used for therapeutic applications, anantigen binding protein can inhibit, interfere with or modulate one ormore biological activities of a PCSK9 (eg, one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form). Inone embodiment, ligand binds specifically to human PCSK9 (eg, one ormore of the rare variants disclosed herein and optionally also the aand/or a′ form) and/or substantially inhibits binding of human PCSK9(eg, said one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form) to LDLR by at least 20%, eg,20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuringbinding in an in vitro competitive binding assay). In an example, theligand is an antibody.

In an embodiment, the ligand has a Kd of less (binding more tightly)than 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M for binding to one,two or more of the rare variants disclosed herein and optionally alsothe a and/or a′ form. In an example, Kd is determined using SPR.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to one or more of the rare PCSK9 variants disclosed herein (andoptionally also the a and/or a′ form) of less than 1 microM, 1000 nM to100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10pM, 10 pM to 1 pM.

In an embodiment, the ligand has an IC50 for blocking the binding ofLDLR to the a and/or a′ form of PCSK9 that is no more than 1000, 100,90, 80, 70, 60, 50, 40, 30, 20 or 10-fold more (ie, more inhibitory)than the IC50 for blocking the binding of LDLR to one or more of therare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteinscomprising a sequence selected from SEQ ID NOs: 4 to 27). Additionallyor alternatively, for example, the ligand has an IC50 for blocking thebinding of LDLR to (i) the a and/or a′ form of less than 1 microM, 1000nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pMto 200 pM, less than 200 pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pMto 10 pM, 10 pM to 1 pM, eg, in the range of 1 mM to 1 pM (eg, 1 mM to100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM) and (ii) oneor more PCSK9 proteins comprising a sequence selected from SEQ ID NOs: 4to 27 of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nMto 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pMto 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM, eg, in therange of 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10pM; or 100 pM to 1 pM).

In an embodiment, the ligand binds to the a and/or a′ form of PCSK9 witha binding affinity (Kd) that is greater than up to 10%, greater than upto 20%, greater than up to 40%, greater than up to 50%, greater than upto 55%, greater than up to 60%, greater than up to 65%, greater than upto 70%, greater than up to 75%, greater than up to 80%, greater than upto 85%, greater than up to 90%, greater than up to 95% or greater thanup to 100% (ie, is double) relative to binding to a PCSK9 comprising asequence selected from SEQ ID NOs: 4 to 27. Such binding measurementscan be made using a variety of binding assays known in the art, eg,using surface plasmon resonance (SPR), such as by Biacore™ or using theProteOn XPR36™ (Bio-Rad®), or using KinExA® (Sapidyne Instruments, Inc).

In one embodiment, the surface plasmon resonance (SPR) is carried out at25° C. In another embodiment, the SPR is carried out at 37° C.

In one embodiment, the SPR is carried out at physiological pH, such asabout pH7 or at pH7.6 (eg, using Hepes buffered saline at pH7.6 (alsoreferred to as HBS-EP)).

In one embodiment, the SPR is carried out at a physiological salt level,eg, 150 mM NaCl.

In one embodiment, the SPR is carried out at a detergent level of nogreater than 0 05% by volume, eg, in the presence of P20 (polysorbate20; eg, Tween-20™) at 0.05% and EDTA at 3 mM.

In one example, the SPR is carried out at 25° C. or 37° C. in a bufferat pH7.6, 150 mM NaCl, 0.05% detergent (eg, P20) and 3 mM EDTA. Thebuffer can contain 10 mM Hepes. In one example, the SPR is carried outat 25° C. or 37° C. in HBS-EP. HBS-EP is available from Teknova Inc(California; catalogue number H8022).

In an example, the affinity of the ligand which is an antibody isdetermined using SPR by

1. Coupling anti-mouse (or other relevant vertebrate) IgG (eg, BiacoreBR-1008-38) to a biosensor chip (eg, GLM chip) such as by primary aminecoupling;

2. Exposing the anti-mouse IgG (vertebrate antibody) to a test IgGantibody to capture test antibody on the chip;

3. Passing the test antigen over the chip's capture surface at 1024 nM,256 nM, 64 nM, 16 nM, 4 nM with a 0 nM (i.e. buffer alone); and

4. And determining the affinity of binding of test antibody to testantigen using surface plasmon resonance, eg, under an SPR conditiondiscussed above (eg, at 25° C. in physiological buffer). SPR can becarried out using any standard SPR apparatus, such as by Biacore™ orusing the ProteOn XPR36™ (Bio-Rad®).

Regeneration of the capture surface can be carried out with 10 mMglycine at pH1.7. This removes the captured antibody and allows thesurface to be used for another interaction. The binding data can befitted to 1:1 model inherent using standard techniques, eg, using amodel inherent to the ProteOn XPR36™ analysis software.

In an embodiment, assaying or testing of a ligand of the invention iscarried out at or substantially at pH7 (eg, for in vitro tests andassays) and at or substantially at rtp.

One example of an IgG2 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 154, FIG. 3KK of US20120093818A1, which sequence isincorporated herein by reference.

One example of an IgG4 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 155, FIG. 3KK of US20120093818A1, which sequence isincorporated herein by reference.

One example of a kappa light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG.3KK which sequence is incorporated herein by reference.

One example of a lambda light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG.3KK of US20120093818A1, which sequence is incorporated herein byreference.

In examples of the present invention, the ligand binds mature PCSK9, eg,a mature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the catalyticdomain of PCSK9, eg, of a mature form of one or more of the rarevariants disclosed herein and optionally also the a and/or a′ form.

In examples of the present invention, the ligand binds the prodomain ofPCSK9, eg, of a mature form of one or more of the rare variantsdisclosed herein and optionally also the a and/or a′ form.

In some embodiments, the ligand binds to the V domain of PCSK9, eg, of amature form of one or more of the rare variants disclosed herein andoptionally also the a and/or a′ form. In some embodiments, the ligandbinds to the V domain of PCSK9 (eg, of a mature form of one or more ofthe rare variants disclosed herein and optionally also the a and/or a′form) and prevents (or reduces, eg, by at least 10%) PCSK9 from bindingto LDLR. In some embodiments, the ligand binds to the V domain of PCSK9(eg, of a mature form of one or more of the rare variants disclosedherein and optionally also the a and/or a′ form), and while it does notprevent (or reduce) the binding of PCSK9 to LDLR, the ligand prevents orreduces (eg, by at least 10%) the adverse activities mediated throughPCSK9 on LDLR.

In examples of the present invention, the ligand is or comprises a fullyhuman antibody. In an example, the ligand comprises human variableregions or humanised variable regions.

In an example, the ligand of the invention specifically binds to anepitope of (i) a PCSK9 of said alternative or (ii) a human PCSK9selected from the group consisting of forms f c, r, p, m, e, h, aj andq, wherein the epitope comprises at least one amino acid that is notfound in form a. For example, the amino acid is selected from the groupconsisting of 46L, 53V, 425S, 443T, 474V, 619P and 670G (numbering asused in SEQ ID NO:1). For example, the amino acid is selected from thegroup consisting of 425S, 443T, 474V, 619P and 670G (numbering as usedin SEQ ID NO:1). For example, the amino acid is selected from the groupconsisting of 425S and 443T (numbering as used in SEQ ID NO:1). Forexample, the amino acid is selected from the group consisting of 474V,619P and 670G (numbering as used in SEQ ID NO:1). In an example, thePCSK9 form is the mature form. In an example, the PCSK9 form is thepro-form. In an example, the ligand also specifically binds to form aand/or a′. In an embodiment, the ligand specifically binds to an epitopeof form f PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to an epitope of form c PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to an epitope of form r PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to an epitope of form pPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to anepitope of form m PCSK9, wherein the epitope comprises at least oneamino acid that is not found in form a. In an embodiment, the ligandspecifically binds to an epitope of form e PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to an epitope of form h PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to an epitopeof form aj PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to an epitope of form q PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the pro-domain of a humanPCSK9 selected from the group consisting of forms f c, r, p, m, e, h, ajand q. In an example, the ligand also specifically binds to thepro-domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the pro-domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform c PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the pro-domain of form r PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the pro-domain of form p PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform m PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the pro-domain of form e PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the pro-domain of form h PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the pro-domain ofform aj PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the pro-domain of form q PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the catalytic domain of ahuman PCSK9 selected from the group consisting of forms f c, r, p, m, e,h, aj and q. In an example, the ligand also specifically binds to thecatalytic domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the catalytic domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the catalytic domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the catalytic domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the catalytic domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the catalytic domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to thecatalytic domain of form h PCSK9, wherein the epitope comprises at leastone amino acid that is not found in form a. In an embodiment, the ligandspecifically binds to the catalytic domain of form aj PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the catalytic domainof form q PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a.

In an embodiment, ligand binds specifically to the C-terminal domain ofa human PCSK9 selected from the group consisting of forms f c, r, p, m,e, h, aj and q. In an example, the ligand also specifically binds to theC-terminal domain of form a and/or a′. In an embodiment, the ligandspecifically binds to the C-terminal domain of form f PCSK9, wherein theepitope comprises at least one amino acid that is not found in form a.In an embodiment, the ligand specifically binds to the C-terminal domainof form c PCSK9, wherein the epitope comprises at least one amino acidthat is not found in form a. In an embodiment, the ligand specificallybinds to the C-terminal domain of form r PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the C-terminal domain ofform p PCSK9, wherein the epitope comprises at least one amino acid thatis not found in form a. In an embodiment, the ligand specifically bindsto the C-terminal domain of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the C-terminal domain of form e PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form h PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a. In an embodiment, theligand specifically binds to the C-terminal domain of form aj PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theC-terminal domain of form q PCSK9, wherein the epitope comprises atleast one amino acid that is not found in form a.

In an embodiment, ligand binds specifically to the substrate-bindinggroove of a human PCSK9 selected from the group consisting of forms f c,r, p, m, e, h, aj and q (see Cunningham et al., Nat Struct Mol Biol.2007 May; 14(5):413-9. Epub 2007 Apr. 15, “Structural and biophysicalstudies of PCSK9 and its mutants linked to familialhypercholesterolemia”, incorporated herein in its entirety byreference). In an example, the ligand also specifically binds to thesubstrate-binding groove of form a and/or a′. In an embodiment, theligand specifically binds to the Substrate-binding groove of formfPCSK9, wherein the epitope comprises at least one amino acid that isnot found in form a. In an embodiment, the ligand specifically binds tothe Substrate-binding groove of form c PCSK9, wherein the epitopecomprises at least one amino acid that is not found in form a. In anembodiment, the ligand specifically binds to the Substrate-bindinggroove of form r PCSK9, wherein the epitope comprises at least one aminoacid that is not found in form a. In an embodiment, the ligandspecifically binds to the Substrate-binding groove of form p PCSK9,wherein the epitope comprises at least one amino acid that is not foundin form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form m PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form ePCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form h PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a. In an embodiment,the ligand specifically binds to the Substrate-binding groove of form ajPCSK9, wherein the epitope comprises at least one amino acid that is notfound in form a. In an embodiment, the ligand specifically binds to theSubstrate-binding groove of form q PCSK9, wherein the epitope comprisesat least one amino acid that is not found in form a.

Reference is made to US20120093818A1 (Amgen, Inc), the entire disclosureof which is incorporated herein. This patent application disclosesrelevant ligands for use in the present invention, as well as examplesand methods of producing and testing ligands that can be used withreference to the present invention.

In an example, the ligand is or comprises an antibody disclosed in Table2 of US20120093818A1 (Amgen, Inc) or is a PCSK9-binding derivativethereof

In an embodiment, the PCSK9-binding ligand of the invention is selectedfrom the antigen binding proteins disclosed in US20120093818A1 (Amgen,Inc), eg, in paragraphs [0009] to and [0058] to [0063] ofUS20120093818A1; all of these disclosures (including the sequences ofsuch proteins) are incorporated herein by reference as though explicitlyrecited herein and for possible inclusion in one or more claims or foruse in the present invention.

In this paragraph SEQ ID NOs are those as appearing in US20120093818A1(Amgen, Inc) and these sequences are incorporated herein by reference asthough explicitly recited herein and for possible inclusion in one ormore claims or for use in the present invention. In some aspects, theligand of the invention comprises an isolated antigen binding proteinthat binds PCSK9 comprising: A) one or more heavy chain complementarydetermining regions (CDRHs) selected from the group consisting of: (i) aCDRH1 from a CDRH1 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii)a CDRH2 from a CDRH2 in a sequence selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60;(iii) a CDRH3 from a CDRH3 in a sequence selected from the groupconsisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54,55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,and 60; and (iv) a CDRH of (i), (ii), and (iii) that contains one ormore amino acid substitutions, deletions or insertions of no more than 4amino acids; B) one or more light chain complementary determiningregions (CDRLs) selected from the group consisting of: (i) a CDRL1 froma CDRL1 in a sequence selected from the group consisting of SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from aCDRL2 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a CDRL3 from aCDRL3 in a sequence selected from the group consisting of SEQ ID NO: 5,7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv) a CDRL of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 4 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B). In someembodiments, the isolated antigen binding protein comprises at least oneCDRH of A) and at least one CDRL of B). In some embodiments, theisolated antigen binding protein comprises at least two CDRH of A) andat least two CDRL of B). In some embodiments, the isolated antigenbinding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 andCDRL3. In some embodiments, the CDRH of A) is selected from at least oneof the group consisting of: (i) a CDRH1 amino acid sequence selectedfrom the CDRH1 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acid sequence selectedfrom the CDRH2 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acid sequence selectedfrom the CDRH3 in a sequence selected from the group consisting of SEQID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids. In addition, the CDRL of B) is selectedfrom at least one of the group consisting of: (i) a CDRL1 amino acidsequence selected from the CDRL1 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2 amino acidsequence selected from the CDRL2 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a CDRL3 amino acidsequence selected from the CDRL3 in a sequence selected from the groupconsisting of SEQ ID NO: 12, 35, 32, and 23; and (iv) a CDRL of (i),(ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B. In someembodiments, the CDRH of A) is selected from at least one of the groupconsisting of: (i) a CDRH1 amino acid sequence of the CDRH1 amino acidsequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequenceof the CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of(i), (ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids; said CDRL of B)is selected from at least one of the group consisting of: (i) a CDRL1amino acid sequence of the CDRL1 amino acid sequence in SEQ ID NO: 12;(ii) a CDRL2 amino acid sequence of the CDRL2 amino acid sequence in SEQID NO: 12; (iii) a CDRL3 amino acid sequence of the CDRL3 amino acidsequence in SEQ ID NO: 12; and (iv) a CDRL of (i), (ii) and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 2 amino acids; or C) one or more heavy chain CDRHs of A)and one or more light chain CDRLs of B). In some embodiments, theantigen binding protein comprises A) a CDRH1 of the CDRH1 sequence inSEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and aCDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) a CDRL1 of theCDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2 sequence in SEQ IDNO: 12, and a CDRL3 of the CDRL3 sequence in SEQ ID NO: 12. In someembodiments, the antigen binding protein comprises a heavy chainvariable region (VH) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 74, 85,71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62,89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or a light chainvariable region (VL) having at least 80% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, the VHhas at least 90% sequence identity with an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, and 60, and/or the VL has at least 90% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In someembodiments, the VH is selected from the group consisting of SEQ ID NO:74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91,64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL isselected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36,37, 38, 39, 40, 42, 44, and 46.

In an example of any aspect of the invention, the PCSK9-targeting orbinding ligand comprises or consists of AMG145 or 31H4, 16F12, 11F1, 8A3or 21B12 disclosed in US20120093818A1 (Amgen, Inc) or an antibodycomprising the variable domains of AMG145, 31H4, 16F12, 11F1, 8A3 or21B12, the disclosures of which (including sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention.Preferably, the PCSK9-targeting or binding ligand comprises or consistsof AMG145.

In an example, the AMG145 or other ligand of the invention isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). In an example, the ligand of the invention is produced inCHO.

Reference is made to US20110065902A1 (Regeneron Pharmaceuticals, Inc),the entire disclosure of which is incorporated herein. This patentapplication discloses relevant ligands for use in the present invention,as well as examples and methods of producing and testing ligands anddetermining medical efficacy that can be used with reference to thepresent invention.

Reference is made to the following PCT applications, the entiredisclosures of which are incorporated herein. These disclose relevantligands for use in the present invention, as well as examples andmethods of producing and testing ligands and determining medicalefficacy that can be used with reference to the present invention.

WO2008057457

WO2008057458

WO2008057459

WO2008063382

WO2008133647

WO2009100297

WO2009100318

WO2011037791

WO2011053759

WO2011053783

WO2008125623

WO2011072263

WO2009055783

WO2010029513

WO2011111007

WO2010077854

Antibody ligands to PCSK9 are described in, for example, WO 2008/057457,WO 2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US2008/0008697; each of which is incorporated by reference herein in itsentirety.

In an example, the ligand is or comprises an antibody disclosed in theExamples of US20110065902A1 (eg, 316P or 300N) or is a PCSK9-bindingderivative thereof. All of these disclosures (including the sequences ofsuch proteins and corresponding nucleotide sequences) are incorporatedherein by reference as though explicitly recited herein and for possibleinclusion in one or more claims or for use in the present invention. Inan embodiment, the ligand is or comprises the variable domains ofantibody 316P or 300N disclosed in US20110065902A1 or is (or comprises)such antibody or a PCSK9-binding derivative thereof

In an embodiment, the ligand is or comprises the variable domains ofantibody alirocumab or SAR236553/REGN727 (Sanofi Aventis/Regeneron) oris (or comprises) such antibody or a PCSK9-binding derivative thereof.In an example, the alirocumab is glycosylated, eg, has humanglycosylation (eg, produced by a CHO, Cos or Hek293 cell). Preferably,the ligand is alirocumab or SAR236553/REGN727.

In an embodiment, the ligand is or comprises the variable domains ofantibody evolocumab or or is (or comprises) such antibody or aPCSK9-binding derivative thereof. In an example, the antibody isglycosylated, eg, has human glycosylation (eg, produced by a CHO, Cos orHek293 cell). Preferably, the ligand is evolocumab.

In an embodiment, the ligand is selected from evolocumab, 1D05-IgG2(Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat) and alirocumab.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2011/0065902, incorporated herein byreference):—

1. An antibody or antigen-binding fragment thereof which specificallybinds hPCSK9, wherein the antibody or antigen-binding fragment comprisesthe heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pairhaving SEQ ID NOs: 218/226.

2. The antibody or antigen-binding fragment of concept 1 comprisingheavy and light chain CDR amino acid sequences having SEQ ID NOs: 220,222, 224, 228, 230 and 232.

3. The antibody or antigen-binding fragment of concept 2 comprising anHCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR havingthe amino acid sequence of SEQ ID NO: 226.

4. An antibody or antigen-binding fragment thereof which binds to thesame epitope on hPCSK9 as an antibody comprising heavy and light chainCDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and232.

5. An antibody or antigen-binding fragment thereof which competes forbinding to hPCSK9 with an antibody comprising heavy and light chain CDRamino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

In an embodiment, the ligand is selected from the following (sequencesand definitions as per US2012/0093818, incorporated herein byreference):—

1. An isolated neutralizing antigen binding protein that binds to aPCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1,wherein the neutralizing antigen binding protein decreases the LDLRlowering effect of PCSK9 on LDLR, wherein the antigen binding proteincomprises a light chain comprising an amino acid sequence of SEQ ID NO:46, and wherein the antigen binding protein comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO: 60.2. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR non-competitiveneutralizing antigen binding protein.3. The isolated neutralizing antigen binding protein of concept 2,wherein the antigen binding protein is a LDLR competitive neutralizingantigen binding protein.4. An antigen binding protein that selectively binds to PCSK9, whereinsaid antigen binding protein binds to PCSK9 with a Kd that is less than100 pM.5. An antigen binding protein that binds to a PCSK 9 protein of SEQ IDNO: 303 in a first manner, wherein the antigen binding protein binds toa variant of PCSK9 in a second manner, wherein said PCSK9 variant has atleast one point mutation at a position selected from the groupconsisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390,413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 ofSEQ ID NO: 303, wherein the first manner comprises a first EC50, a firstBmax, or a first EC50 and a first Bmax, wherein the second mannercomprises a second EC50, a second Bmax, or a second EC50 and a secondBmax, and wherein a value for the first manner is different from a valuefor the second manner, and wherein the antigen binding protein comprisesa light chain comprising an amino acid sequence of SEQ ID NO: 46, andwherein the antigen binding protein comprises a heavy chain comprisingan amino acid sequence of SEQ ID NO: 60.6. The antigen binding protein of concept 6, wherein the first mannercomprises a first Bmax, wherein the second manner comprises a secondBmax that is different from the first Bmax, and wherein said PCSK9variant has at least one point mutation selected from the groupconsisting of: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R,R519E, H521R, and Q554R.7. The antigen binding protein of concept 6, wherein the antigen bindingprotein binds to PCSK9 at a location that overlaps with a location thatLDLR binds to PCSK9.8. A method of making an antigen binding protein that binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theantigen binding protein decreases the LDLR lowering effect of PCSK9 onLDLR, said method comprising: providing a host cell comprising a nucleicacid sequence that encodes the antigen binding protein; and maintainingthe host cell under conditions in which the antigen binding protein isexpressed, wherein the antigen binding protein comprises a light chaincomprising an amino acid sequence of SEQ ID NO: 46, and wherein theantigen binding protein comprises a heavy chain comprising an amino acidsequence of SEQ ID NO: 60.9. A method for treating or preventing a condition associated withelevated serum cholesterol levels in a subject, said method comprisingadministering to a subject in need thereof an effective amount of anisolated neutralizing antigen binding protein simultaneously orsequentially with an agent that elevates the availability of LDLRprotein, wherein the isolated antigen binding protein binds to a PCSK9protein comprising the amino acid sequence of SEQ ID NO: 1, wherein theneutralizing antigen binding protein decreases the LDLR lowering effectof PCSK9 on LDLR, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.10. The method of concept 10, wherein the agent that elevates theavailability of LDLR protein comprises a statin.11. An antigen binding protein that binds to PCSK9, wherein when theantigen binding protein is bound to PCSK9, the antibody is positioned 8angstroms or less from at least one of the following residues of PCSK9:S153, S188, 1189, Q190, S191, D192, R194, E197, G198, R199, V200, D224,R237, D238, K243, S373, D374, S376, T377, F379, 1154, T187, H193, E195,1196, M201, V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,C375, or C378, wherein the antigen binding protein comprises a lightchain comprising an amino acid sequence of SEQ ID NO: 46, and whereinthe antigen binding protein comprises a heavy chain comprising an aminoacid sequence of SEQ ID NO: 60.

The ligand can be used for the treatment, therapy, prophylaxis and/ordiagnosis of one or more diseases or conditions or susceptibilitythereto, wherein such diseases or conditions comprise those disclosed inUS20120093818A1 (Amgen, Inc) and US20110065902A1 (RegeneronPharmaceuticals, Inc), eg, a disease or condition disclosed inparagraphs [0375] to [0383] of US20120093818A1, which disclosure isincorporated herein by reference in its entirety for inclusion in onemore claims herein.

The ligand can be administered to a human characterised as described inUS20120093818A1 (Amgen, Inc) or US20110065902A1.

The ligand can be administered in a form or combination disclosed inUS20120093818A1 (Amgen, Inc) or US20110065902A1, which disclosure isincorporated herein by reference. For example, the ligand with a drug,excipient, diluent or carrier as described in US20120093818A1 (Amgen,Inc) or US20110065902A1 (eg, as disclose in paragraphs [0384] to [0412]of US20120093818A1), which disclosure is incorporated herein byreference, and the present invention also relates to the correspondingpharmaceutical compositions comprising the combination of a ligand ofthe invention and such a further agent.

The ligand can be used in a method of diagnosis as set out inUS20120093818A1 (Amgen, Inc) or US20110065902A1, eg, in paragraphs[0413] to [0415] of US20120093818A1 which disclosure is incorporatedherein by reference.

Diagnostic Applications

In some embodiments, the ligand of the invention is a diagnostic tool.The ligand can be used to assay the amount of PCSK9 present in a sampleand/or subject. As will be appreciated by one of skill in the art, suchligands need not be neutralizing ligands. In some embodiments, thediagnostic ligand is not a neutralizing ligand. In some embodiments, thediagnostic ligand binds to a different epitope than a neutralizingligand binds to. In some embodiments, the two ligands do not competewith one another.

In some embodiments, the ligands of the invention are used or providedin an assay kit and/or method for the detection of PCSK9 in mammaliantissues or cells in order to screen/diagnose for a disease or disorderassociated with changes in levels of PCSK9. The kit comprises a ligandthat binds PCSK9 and means for indicating the binding of the ligand withPCSK9, if present, and optionally PCSK9 protein levels. Various meansfor indicating the presence of a ligand can be used. For example,fluorophores, other molecular probes, or enzymes can be linked to theligand and the presence of the ligand can be observed in a variety ofways. The method for screening for such disorders can involve the use ofthe kit, or simply the use of one of the disclosed ligands and thedetermination of whether the ligand binds to PCSK9 in a sample. As willbe appreciated by one of skill in the art, high or elevated levels ofPCSK9 will result in larger amounts of the ligand binding to PCSK9 inthe sample. Thus, degree of ligand binding can be used to determine howmuch PCSK9 is in a sample. Subjects or samples with an amount of PCSK9that is greater than a predetermined amount (e.g., an amount or rangethat a person without a PCSK9 related disorder would have) can becharacterized as having a PCSK9 mediated disorder. In some embodiments,the invention provides a method wherein the ligand is administered to asubject taking a statin, in order to determine if the statin hasincreased the amount of PCSK9 in the subject.

In some embodiments, the ligand is a non-neutralizing ligand and is usedto determine the amount of PCSK9 in a subject receiving an ABP and/orstatin treatment.

In some embodiments, the ligand of the invention can specifically bindhuman PCSK9 (eg, one, two or more rare variant forms disclosed herein)and is characterized by at least one of: (i) capable of reducing serumtotal cholesterol at least about 25-35% and sustaining the reductionover at least a 24 day period relative to a predose level; (ii) capableof reducing serum LDL cholesterol at least about 65-80% and sustainingthe reduction over at least a 24 day period relative to a predose level;(iii) capable of reducing serum LDL cholesterol at least about 40-70%and sustaining the reduction over at least a 60 or 90 day periodrelative to a predose level; (iv) capable of reducing serum triglycerideat least about 25-40% relative to predose level; (v) does not reduceserum HDL cholesterol or reduces serum HDL cholesterol no more than 5%relative to predose level. In some embodiments, an isolated nucleic acidmolecule is provided and it encodes the ligand. In some embodiments anexpression vector is provided and comprises the nucleic acid molecule.In some embodiments, a pharmaceutical composition is provided and it cancomprise the ligand and a pharmaceutically acceptable carrier. In someembodiments, a method is provided for treating a disease or conditionwhich is ameliorated, improved, inhibited or prevented with a PCSK9antagonist ligand of the invention. The method can compriseadministering a therapeutic amount of the pharmaceutical composition orligand to a subject in need thereof. In some embodiments, the subject isa human subject suffering from hypercholesterolemia, hyperlipidemia,indicated for LDL apheresis, identified as heterozygous for FamilialHypercholesterolemia, statin intolerant, statin uncontrolled, at riskfor developing hypercholesterolemia, dyslipidemia, cholestatic liverdisease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosisand cardiovascular diseases. In some embodiments, a method of providinga treatment or therapy is provided to a subject. In some embodiments,the method comprises reducing serum cholesterol at least about 40-70%over at least 60 to 90 days. In some embodiments, a method of receivingtreatment or therapy is provided, the method can comprise receiving aligand thereof at a frequency of once every 60 to 90 days.

In one aspect, the invention provides a ligand of the invention which isor comprises an human antibody or antigen-binding fragment of a humanantibody that specifically binds and inhibits human proproteinconvertase subtilisin/kexin type 9 (hPCSK9, eg, one, two or more rarevariant forms disclosed herein and optionally form a and/or form a′),characterized by the ability to reduce serum LDL cholesterol in a humanby 40-80% over a 24, 60 or 90 day period relative to predose levels,with little or no reduction in serum HDL cholesterol and/or with littleor no measurable effect on liver function, as determined by ALT and ASTmeasurements.

In one embodiment, the ligand of the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum total cholesterol at least about 25-35%and sustaining the reduction over at least a 24 day period relative to apredose level, preferably the reduction in serum total cholesterol is atleast about 30-40%;

(ii) capable of reducing serum LDL cholesterol at least about 65-80% andsustaining the reduction over at least a 24 day period relative to apredose level;

(iii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;

(iv) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

See US2011/0065902 for definitions of these terms and optional features,the disclosure of which is incorporated herein by reference in itsentirety.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum LDL cholesterol at least about 40-70% andsustaining the reduction over at least a 60 or 90 day period relative toa predose level;

(ii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;

(iii) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as exhibiting an enhanced binding affinity (KD) for hPCSK9at pH 5.5 relative to the KD at pH 7.4, as measured by plasmon surfaceresonance In a specific embodiment, the antibody or fragment thereofexhibits at least a 20-fold, at least a 40-fold or at least a 50-foldenhanced affinity for PCSK9 at an acidic pH relative to a neutral pH, asmeasured by surface plasmon resonance

In one embodiment, the antibody or antigen-binding fragment ischaracterized as not exhibiting an enhanced binding affinity for PCSK9at an acidic pH relative to a neutral pH, as measured by surface plasmonresonance In a specific embodiment, the antibody or fragment thereofexhibits a decreased binding affinity at an acidic pH.

In another embodiment, the antibody or antigen-binding fragment bindshuman, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey,mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen-binding fragment binds humanand monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising one or more of aheavy chain variable region (HCVR), light chain variable region (LCVR),HCDR1, HCDR2, HCDR3 disclosed in any of paragraphs [023]-[037] ofUS2011/0065902, the disclosures of which are incorporated herein byreference.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds hPCSK9,wherein the antibody or fragment comprises heavy and light chain CDRdomains contained within heavy and light chain sequence pairs selectedfrom the group consisting of SEQ ID NO (using the sequence numbering inU52011/0065902): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68,70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140,142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202,210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264,266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332,334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394,402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456,458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524,526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586,594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648,650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706, 714/716,718/720, 722/730, 738/740 and 742/744. In one embodiment, the CDRsequences are contained within HCVR and LCVR selected from the aminoacid sequence pairs of SEQ ID NO: 50/58, 66/68, 70/72, 74/82, 90/92,94/96, 122/130, 138/140, 142/144, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 314/322, 330/332 and 334/336. In more specificembodiments, the CDR sequences are comprised within HCVR/LCVR sequencesselected from SEQ ID NO: 90/92 or 218/226.

In an example, the invention features a pharmaceutical compositioncomprising a ligand of the invention, wherein the ligand comprises orconsists of a recombinant human antibody or fragment thereof whichspecifically binds hPCSK9 and a pharmaceutically acceptable carrier. Inone embodiment, the invention features a composition which is acombination of a ligand of the invention (eg, an antibody orantigen-binding fragment of an antibody), and a second therapeuticagent. The second therapeutic agent may be any agent that isadvantageously combined with the ligand of the invention, for example,an agent capable of inducing a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as, for example, cerovastatin, atorvastatin,simvastatin, pitavastin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc; capable of inhibiting cholesterol uptake and or bileacid re-absorption; capable of increasing lipoprotein catabolism (suchas niacin); and/or activators of the LXR transcription factor that playsa role in cholesterol elimination such as 22-hydroxycholesterol.

In an example, the invention provides a method for inhibiting hPCSK9activity using the anti-PCSK9 ligand of the invention (eg, an antibodyor antigen-binding portion of the antibody of the invention), whereinthe therapeutic methods comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment of an antibody of the invention. Thedisorder treated is any disease or condition which is improved,ameliorated, inhibited or prevented by removal, inhibition or reductionof PCSK9 activity. Specific populations treatable by the therapeuticmethods of the invention include subjects indicated for LDL apheresis,subjects with PCSK9-activating mutations (gain of function mutations,“GOF”), subjects with heterozygous Familial Hypercholesterolemia (heFH);subjects with primary hypercholesterolemia who are statin intolerant orstatin uncontrolled; and subjects at risk for developinghypercholesterolemia who may be preventably treated. Other indicationsinclude dyslipidemia associated with secondary causes such as Type 2diabetes mellitus, cholestatic liver diseases (primary biliarycirrhosis), nephrotic syndrome, hypothyroidism, obesity; and theprevention and treatment of atherosclerosis and cardiovascular diseases.

In specific embodiments of the method of the invention, the ligand ofthe invention (eg, anti-hPCSK9 antibody or antibody fragment of theinvention) is useful to reduce elevated total cholesterol, non-HDLcholesterol, LDL cholesterol, and/or apolipoprotein B (apolipoproteinB100).

The ligand (eg, antibody or antigen-binding fragment) of the inventionmay be used alone or in combination with a second agent, for example, anHMG-CoA reductase inhibitor and/or another lipid lowering drug.

Treatment Population

The invention provides therapeutic methods for treating a human patientin need of a composition or ligand of the invention. While modificationsin lifestyle and conventional drug treatment are often successful inreducing cholesterol levels, not all patients are able to achieve therecommended target cholesterol levels with such approaches. Variousconditions, such as familial hypercholesterolemia (FH), appear to beresistant to lowering of LDL-C levels in spite of aggressive use ofconventional therapy. Homozygous and heterozygous familialhypercholesterolemia (hoFH, heFH) is a condition associated withpremature atherosclerotic vascular disease. However, patients diagnosedwith hoFH are largely unresponsive to conventional drug therapy and havelimited treatment options. Specifically, treatment with statins, whichreduce LDL-C by inhibiting cholesterol synthesis and upregulating thehepatic LDL receptor, may have little effect in patients whose LDLreceptors are non-existent or defective. A mean LDL-C reduction of onlyless than about 20% has been recently reported in patients withgenotype-confirmed hoFH treated with the maximal dose of statins. Theaddition of ezetimibe 10 mg/day to this regimen resulted in a totalreduction of LDL-C levels of 27%, which is still far from optimal.Likewise, many patients are statin non-responsive, poorly controlledwith statin therapy, or cannot tolerate statin therapy; in general,these patients are unable to achieve cholesterol control withalternative treatments. There is a large unmet medical need for newtreatments that can address the short-comings of current treatmentoptions.

Specific populations treatable by the therapeutic methods of theinvention include patients indicated for LDL apheresis, subjects withPCSK9-activating (GOF) mutations, heterozygous FamilialHypercholesterolemia (heFH); subjects with primary hypercholesterolemiawho are statin intolerant or statin uncontrolled; and subjects at riskfor developing hypercholesterolemia who may be preventably treated.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-PCSK9 ligands, antibodies or antigen-binding fragments thereof ofthe present invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the ligand, eg, antibody, of the present invention isused for treating various conditions and diseases associated with PCSK9,including hypercholesterolemia, disorders associated with LDL andapolipoprotein B, and lipid metabolism disorders, and the like, in anadult patient, it is advantageous to intravenously administer the ligandor antibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, thus the compositioninvention provides the ligand by e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al.(1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecomposition may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

The invention provides therapeutic methods in which the ligand, eg,antibody or antibody fragment, of the invention is useful to treathypercholesterolemia associated with a variety of conditions involvinghPCSK9. The anti-PCSK9 ligands, eg, antibodies or antibody fragments, ofthe invention are particularly useful for the treatment ofhypercholesterolemia and the like. Combination therapies may include theanti-PCSK9 ligand of the invention with, for example, one or more of anyagent that (1) induces a cellular depletion of cholesterol synthesis byinhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase,such as cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin; (2) inhibitscholesterol uptake and or bile acid re-absorption; (3) increaselipoprotein catabolism (such as niacin); and activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol or fixed combinations such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam), a fixed combination of niacin plus a statin(e.g., niacin with lovastatin); or with other lipid lowering agents suchas omega-3-fatty acid ethyl esters (for example, omacor).

Tailoring Antibodies to Rare PCSK9 Variant Profile

As outline above, the invention includes the possibility to tailortreatment of humans further by selecting antibody-based ligands withvariable domains based on gene segments commonly found in humans of theethnic populations where the variant PCSK9 forms are found to meet theselection criteria of the invention. An example is provided below forligands comprising antibody VH domains derived from recombination ofhuman VH3-23.

The inventor analysed the frequencies and distribution of various humanVH3-23 alleles and realised the desirability of using ligands based onhuman VH3-23 alleles comprising SNP rs56069819. This SNP corresponds toa change from leucine at position 24 in the encoded protein sequence toa valine at that position (L24V change) and the SNP is at coordinate106268889 on human chromosome 14.

FIG. 2 shows the cumulative allele frequency distribution across the1000 Genomes Project database of human VH3-23 alleles comprising SNPrs56069819 (such alleles denonted “C” and the most frequent allele(which does not comprise this SNP) denoted “A”). The figure shows thatVH3-23 alleles comprising SNP rs56069819 are present at a cumulativefrequency of 11% across all human ethnic populations taken as a whole,whereas in certain specific human ethnic sub-populations (ASW, LWK, YRI,CEU and GBR) such alleles are present at an above-average cumulativefrequency. Indicated in the figure are those human PCSK9 variant forms(marked “Variants”) that are found in the various sub-populations withabove-average occurrence of human VH3-23 alleles comprising SNPrs56069819. Table 7 shows the VH3-23 variants and the SNPs that theycomprise, as well as their cumulative allele frequencies as found in the1000 Genomes Project database.

Notably, human VH3-23 alleles comprising SNP rs56069819 were found inthe CEU population at a frequency that is almost double the frequency of11% for all populations. For the ASW and YRI populations the frequencywas over a quarter of the population. Thus, the invention advantageouslyenables one to select a ligand comprising an antibody or antibodyfragment, wherein the antibody or fragment comprises a VH domain derivedfrom the recombination of a human VH gene segment, a human D genesegment and a human JH gene segment, the VH gene segment comprising anucleotide sequence that comprises SNP rs56069819 (dbSNP numbering,build number as recited above).

In an example, one can tailor the treatment further by selecting such aligand that specifically binds to a human PCSK9 selected from forms: fc, m, e, h, p, q and aj, such forms being those appearing in humanpopulations ASW, LWK, YRI, CEU and GBR.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation I474V in SEQ ID NO: 1, wherein the human is of ASW, LWK, YRI,CEU or GBR ancestry. Optionally, the human comprises gene segmentVH3-23*04 and/or a nucleotide sequence encoding said PCSK9 comprising amutation I474V in SEQ ID NO: 1. In an example, additionally the ligandis an antibody or fragment comprising a VH domain derived from therecombination of human gene segment VH3-23*04.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation I474V in SEQ ID NO: 1 (eg, form f r, p, e or aj), wherein thehuman is of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the humancomprises gene segment VH3-23*04 and/or a nucleotide sequence encodingsaid PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example,additionally the ligand is an antibody or fragment comprising a VHdomain derived from the recombination of human gene segment VH3-23*04.Optionally, the ligand is for treating or preventing dislipidemia insaid human, eg, for reducing cholesterol or maintaining a previouslyreduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation E670G in SEQ ID NO: 1 (eg, form c, r or q), wherein the humanis of ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the humancomprises gene segment VH3-23*04 and/or a nucleotide sequence encodingsaid PCSK9 comprising a mutation I474V in SEQ ID NO: 1. In an example,additionally the ligand is an antibody or fragment comprising a VHdomain derived from the recombination of human gene segment VH3-23*04.Optionally, the ligand is for treating or preventing dislipidemia insaid human, eg, for reducing cholesterol or maintaining a previouslyreduced cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation Q619P in SEQ ID NO: 1 (eg, form h), wherein the human is ofASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises genesegment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9comprising a mutation I474V in SEQ ID NO: 1. In an example, additionallythe ligand is an antibody or fragment comprising a VH domain derivedfrom the recombination of human gene segment VH3-23*04. Optionally, theligand is for treating or preventing dislipidemia in said human, eg, forreducing cholesterol or maintaining a previously reduced cholesterollevel in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation N425S in SEQ ID NO: 1 (eg, form e), wherein the human is ofASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises genesegment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9comprising a mutation I474V in SEQ ID NO: 1. In an example, additionallythe ligand is an antibody or fragment comprising a VH domain derivedfrom the recombination of human gene segment VH3-23*04. Optionally, theligand is for treating or preventing dislipidemia in said human, eg, forreducing cholesterol or maintaining a previously reduced cholesterollevel in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation R46L in SEQ ID NO: 1 (eg, form aj), wherein the human is ofASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprises genesegment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9comprising a mutation I474V in SEQ ID NO: 1. In an example, additionallythe ligand is an antibody or fragment comprising a VH domain derivedfrom the recombination of human gene segment VH3-23*04. Optionally, theligand is for treating or preventing dislipidemia in said human, eg, forincreasing cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation A53V in SEQ ID NO: 1 (eg, form p or q), wherein the human isof ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprisesgene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9comprising a mutation I474V in SEQ ID NO: 1. In an example, additionallythe ligand is an antibody or fragment comprising a VH domain derivedfrom the recombination of human gene segment VH3-23*04. Optionally, theligand is for treating or preventing dislipidemia in said human, eg, forincreasing cholesterol level in said human.

In an example, the ligand specifically binds to a human PCSK9 comprisinga mutation A443T in SEQ ID NO: 1 (eg, form m or h), wherein the human isof ASW, LWK, YRI, CEU or GBR ancestry. Optionally, the human comprisesgene segment VH3-23*04 and/or a nucleotide sequence encoding said PCSK9comprising a mutation I474V in SEQ ID NO: 1. In an example, additionallythe ligand is an antibody or fragment comprising a VH domain derivedfrom the recombination of human gene segment VH3-23*04. Optionally, theligand is for treating or preventing dislipidemia in said human, eg, forincreasing cholesterol level in said human.

In an example, the VH gene segment is VH3-23*04, which is a commonlyfound variant that comprises SNP rs56069819 in human populations ASW,LWK, YRI, CEU and GBR.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human that expresses a humanPCSK9 selected from forms: f c, m, e, h, p, q and aj.

In an example, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW, LWK, YRI, CEU orGBR ancestry.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of ASW ancestry, whereinthe human expresses a PCSK9 selected from f c, m, e, h, p and q or thehuman comprises a corresponding nucleotide or amino acid sequence as setout in Table 6. Optionally this ligand comprises a VH domain derivedfrom recombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of LWK ancestry, whereinthe human expresses a PCSK9 selected from f c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of YRI ancestry, whereinthe human expresses a PCSK9 selected from f c, m, e and h or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of CEU ancestry, whereinthe human expresses a PCSK9 selected from f c, p and aj or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an embodiment, the ligand is for treating and/or preventing aPCSK9-mediated disease or condition in a human of GBR ancestry, whereinthe human expresses a PCSK9 selected from f, c and p or the humancomprises a corresponding nucleotide or amino acid sequence as set outin Table 6. Optionally this ligand comprises a VH domain derived fromrecombination of human VH3-23*04.

In an example, the ligand is alirocumab.

In other embodiments of any configuration or TOI herein, as explainedmore fully above, the invention provides for ligands which are tailoredto the human recipient's genotype and/or phenotype based on alternativehuman VH gene segments, or on Vκ, Vλ or constant region gene segments(see further Table 9 for representative variants).

For example, the ligand of the invention comprises or consists of anantibody that comprises a VH domain that is derived from therecombination of a human VH gene segment, a human D gene segment and ahuman JH gene segment, wherein the VH gene segment is selected from thegroup consisting of (i) IGHV1-18*01 and the genome of the humancomprises a human IGHV1-18*01 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGHV1-18*01; or (ii) IGVH1-46*01 and the genome of the humancomprises a human IGHV1-46*01 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGHV1-46*01.

For example, the ligand of the invention comprises or consists of anantibody that comprises a VL domain that is derived from therecombination of a human VL gene segment and a human JL gene segment,wherein the VL gene segment is selected from the group consisting of (i)IGKV4-1*01 and the genome of the human comprises a human IGKV4-1*01nucleotide sequence or the human expresses antibodies comprisingvariable domains derived from the recombination of human IGKV4-1*01;(ii) IGLV2-14*01 and the genome of the human comprises a humanIGLV2-14*01 nucleotide sequence or the human expresses antibodiescomprising variable domains derived from the recombination of humanIGLV2-14*01; or (iii) IGKV1-13*02 and the genome of the human comprisesa human IGKV1-13*02 nucleotide sequence or the human expressesantibodies comprising variable domains derived from the recombination ofhuman IGKV1-13*02.

For example, the inventor identified the possibility of addressing therarer IGH-gamma-1 SNPs 204D (observed cumulative frequency of 0.296) and206L (observed cumulative frequency of 0.283) individually or incombination. These residues are part of the CH3 domain, and as such theyform part of antibody Fc regions. Thus, matching of these CH3 variationswith the patient is especially beneficial for reasons as discussedabove. Thus, in this example the ligand of the invention comprises orconsists of an antibody that comprises a human gamma-1 heavy chainconstant region that comprises an Asp corresponding to position 204 ofSEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ ID NO: 42and wherein the genome of the human comprises a gamma-1 heavy chainconstant region nucleotide sequence that encodes such an Asp or Leu orthe human expresses antibodies comprising human gamma-1 constant regionscomprising such an Asp or Leu. An example of such a ligand isalirocumab.

In another example, the inventor identified the possibility ofaddressing IGH-gamma-2 SNPs. This included consideration of Fc regionvariation—in this respect, the inventor focused on positions 161 and 257which are in the Fc region. Thus, in this example the ligand of theinvention comprises or consists of an antibody that comprises a humangamma-2 heavy chain constant region that comprises an amino acidselected from the group consisting of a Pro corresponding to position 72of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44,a Phe corresponding to position 76 of SEQ ID NO: 44, a Val correspondingto position 161 of SEQ ID NO: 44 and an Ala corresponding to position257 of SEQ ID NO: 44; and wherein the genome of the human comprises agamma-2 heavy chain constant region nucleotide sequence that encodessuch a selected amino acid or the human expresses antibodies comprisinghuman gamma-2 constant regions comprising such a selected amino acid. Anexample of such a ligand is evolocumab or bococizumab.

In another example, the inventor addressed human kappa constant regionvariation. Thus, in this example the ligand of the invention comprisesor consists of an antibody that comprises a human kappa light chainconstant region that comprises a Val corresponding to position 84 of SEQID NO: 50 or a Cys corresponding to position 87 of SEQ ID NO: 50; andwherein the genome of the human comprises a kappa light chain constantregion nucleotide sequence that encodes such a Val or Cys or the humanexpresses antibodies comprising human kappa light chain constant regionscomprising such a Val or Cys. An example of such a ligand is alirocumabor bococizumab.

In another example, the inventor addressed human lambda constant regionvariation. Thus, in this example the ligand of the invention comprisesor consists of an antibody that comprises a human IGLC2*01 light chainconstant region; and wherein the genome of the human comprises a humanIGLC2*01 nucleotide sequence or the human expresses antibodiescomprising human light chain IGLC2*01 constant regions. An example ofsuch a ligand is evolocumab.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   -   1. An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof, or (b) targeting        PCSK9 in a human,        -   wherein the antibody or fragment comprises a human gamma            heavy chain constant region that comprises a first amino            acid that is encoded by a human gamma heavy chain constant            region gene segment SNP, and the antibody or fragment            specifically binds a proprotein convertase subtilisin/kexin            type 9 (PCSK9) amino acid sequence that comprises a            C-terminal domain comprising a mutation selected from the            group consisting of I474V, Q619P, N425S and E670G (eg,            selected from I474V and E670G) in SEQ ID NO:1, wherein the            human comprises a nucleotide sequence encoding said PCSK9            amino acid sequence and comprises a human gamma heavy chain            constant region gene segment comprising said SNP, or the            human expresses antibodies comprising human gamma constant            regions comprising said first amino acid.        -   In an embodiment of (b), the antibody or fragment treats or            reduces cholesterol level or maintains previously reduced            cholesterol level in the human.        -   In an alternative, paragraph 1 provides:—        -   An antibody or antibody fragment for use in a method of (a)            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, or (b)            targeting PCSK9 in a human,        -   wherein the antibody or fragment comprises a human gamma-1            heavy chain constant region that comprises an Asp            corresponding to position 204 of SEQ ID NO: 42 or a Leu            corresponding to position 206 of SEQ ID NO: 42, and the            antibody or fragment specifically binds a proprotein            convertase subtilisin/kexin type 9 (PCSK9) amino acid            sequence that comprises a C-terminal domain comprising a            said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO:1,            wherein the human comprises a nucleotide sequence encoding            said amino acid sequence and comprises an IGHG1*01 human            heavy chain constant region gene segment, or the human            expresses antibodies comprising human gamma-1 constant            regions comprising such an Asp and Leu.        -   In an embodiment, said mutation is I474V. In another            embodiment, said mutation is E670G.        -   In another embodiment, said mutation is N425S. In another            embodiment, said mutation is Q619P.        -   In an embodiment of (b), the antibody or fragment treats or            reduces cholesterol level or maintains previously reduced            cholesterol level in the human.        -   An example provides:—        -   An antibody or antibody fragment for use in a method of (a)            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, or (b)            targeting PCSK9 in a human,        -   wherein the antibody or fragment comprises a human gamma-1            heavy chain constant region that comprises an Asp            corresponding to position 204 of SEQ ID NO: 42 and a Leu            corresponding to position 206 of SEQ ID NO: 42, and the            antibody or fragment specifically binds a proprotein            convertase subtilisin/kexin type 9 (PCSK9) amino acid            sequence that comprises a C-terminal domain comprising a            mutation I474V in SEQ ID NO:1, wherein the human comprises a            nucleotide sequence encoding said amino acid sequence and            comprises an IGHG1*01 human heavy chain constant region gene            segment. Optionally, the antibody or fragment comprises an            IGHG1*01 human heavy chain constant region.        -   In an embodiment of (b), the antibody or fragment treats or            reduces cholesterol level or maintains previously reduced            cholesterol level in the human.        -   Another example provides:—        -   An antibody or antibody fragment for use in a method of (a)            reducing cholesterol level or maintaining previously reduced            cholesterol level in a human in need thereof, or (b)            targeting PCSK9 in a human,        -   wherein the antibody or fragment comprises a human gamma-1            heavy chain constant region that comprises an Asp            corresponding to position 204 of SEQ ID NO: 42 and a Leu            corresponding to position 206 of SEQ ID NO: 42, and the            antibody or fragment specifically binds a proprotein            convertase subtilisin/kexin type 9 (PCSK9) amino acid            sequence that comprises a C-terminal domain comprising a            mutation E670G in SEQ ID NO:1, wherein the human comprises a            nucleotide sequence encoding said amino acid sequence and            comprises an IGHG1*01 human heavy chain constant region gene            segment. Optionally, the antibody or fragment comprises an            IGHG1*01 human heavy chain constant region.        -   In an embodiment of (b), the antibody or fragment treats or            reduces cholesterol level or maintains previously reduced            cholesterol level in the human.        -   In an example, said antibody or antibody fragment has been            determined to specifically bind a proprotein convertase            subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal            domain comprising a mutation selected from the group            consisting of I474V, Q619P, N425S and E670G (eg, selected            from I474V and E670G) in SEQ ID NO: 1, wherein the antibody            or fragment comprises a human gamma-1 heavy chain constant            region that comprises an Asp corresponding to position 204            of SEQ ID NO: 42 and a Leu corresponding to position 206 of            SEQ ID NO: 42 and wherein said human comprises (i) an            IGHG1*01 human heavy chain constant region gene segment            and (ii) a nucleotide sequence encoding said proprotein            convertase subtilisin/kexin type 9 (PCSK9) that comprises a            C-terminal domain comprising said mutation (eg, I474V or            E670G) in SEQ ID NO: 1.    -   2. The antibody or antibody fragment of paragraph 1, wherein the        antibody comprises a human gamma-1 heavy chain constant region        that comprises an Asp corresponding to position 204 of SEQ ID        NO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42.    -   3. The antibody or antibody fragment of paragraph 1 or 2,        wherein the antibody comprises an IGHG1*01 human heavy chain        constant region.    -   4. The antibody or antibody fragment of any one of paragraphs 1        to 3, wherein the human has been determined to comprise the        nucleotide sequence that encodes a PCSK9 comprising a C-terminal        domain comprising said mutation (eg, I474V or E670G) in SEQ ID        NO: 1 and/or a proprotein convertase subtilisin/kexin type 9        (PCSK9) variant protein encoded by the nucleotide sequence of        SEQ ID NO: 29 or 30.    -   5. The antibody or antibody fragment of any one of paragraphs 1        to 4, the method comprising the step of determining that the        human comprises the nucleotide sequence that encodes a PCSK9        comprising a C-terminal domain comprising said mutation (eg,        I474V or E670G) and/or a proprotein convertase subtilisin/kexin        type 9 (PCSK9) variant protein comprising said mutation (eg,        I474V or E670G), optionally, wherein the determining step is        performed before administration of the antibody to the human.    -   6. The antibody or antibody fragment of paragraph 5, wherein the        step of determining comprises assaying a biological sample from        the human for a nucleotide sequence encoding the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.    -   7. The antibody or antibody fragment of paragraph 6, wherein the        assaying comprises contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence encoding the PCSK9 that comprises the            C-terminal domain comprising the mutation (eg, I474V or            E670G) in SEQ ID NO: 1 or that specifically hybridizes to an            antisense of said sequence, wherein said nucleic acid            hybridizes to at least one nucleotide present in said            selected sequence which is not present in SEQ ID NO: 28 or            hybridizes to an antisense sequence thereby forming a            complex when at least one nucleotide sequence encoding the            PCSK9 that comprises the C-terminal domain comprising the            mutation (eg, I474V or E670G) in SEQ ID NO: 1 is present;            and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence encoding the PCSK9 that comprises the C-terminal            domain comprising the mutation (eg, I474V or E670G) in SEQ            ID NO: 1 or comprising an antisense sequence of said            contiguous nucleotides, wherein said sequence of contiguous            nucleotides comprises at least one nucleotide present in            said selected sequence which is not present in SEQ ID NO: 28            thereby forming a complex when the nucleotide sequence            encoding the PCSK9 that comprises a C-terminal domain            comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1            is present; and detecting the presence or absence of the            complex, wherein detecting the presence of the complex            determines that the human comprises the PCSK9 that comprises            the C-terminal domain comprising the mutation (eg, I474V or            E670G) in SEQ ID NO: 1.    -   8. The antibody or antibody fragment of paragraph 6 or 7,        wherein the assaying comprises nucleic acid amplification and        optionally one or more methods selected from sequencing, next        generation sequencing, nucleic acid hybridization, and        allele-specific amplification and/or wherein the assaying is        performed in a multiplex format.    -   9. The antibody or antibody fragment of any one of paragraphs 1        to 8, wherein said antibody or antibody fragment is for        administration to a human that is or has been further determined        to be substantially resistant to statin treatment.    -   10. The antibody or antibody fragment of any one of paragraphs 1        to 9, wherein antibody or antibody fragment is for        administration to a human that is receiving or has received        statin treatment or has reduced responsiveness to statin        treatment.    -   11. The antibody or antibody fragment of paragraph 9 or 10,        wherein said antibody or antibody fragment is for administration        to the human separately or simultaneously with said statin        treatment.    -   12. The antibody or antibody fragment of any one of paragraphs 6        to 10, wherein said biological sample comprises serum, blood,        faeces, tissue, a cell, urine and/or saliva of said human.    -   13. The antibody or antibody fragment of any one of paragraphs 1        to 12, wherein said human is indicated as heterozygous for a        nucleotide sequence encoding the PCSK9 C-terminal domain        comprising a said PCSK9 mutation (eg, I474V or E670G),        optionally, wherein said human is further indicated as        comprising the nucleotide sequence of SEQ ID NO: 28, or said        human is indicated as homozygous for a nucleotide sequence        encoding the PCSK9 C-terminal domain comprising a said PCSK9        mutation (eg, I474V or E670G) in SEQ ID NO: 1.    -   14. The antibody or antibody fragment of any one of paragraphs 1        to 13, wherein said human has been diagnosed with at least one        condition selected from a lipid disorder, hyperlipoproteinemia,        hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart        attack, a stroke, coronary heart disease, atherosclerosis,        peripheral vascular disease, claudication (eg, claudication        associated with elevated cholesterol) and high blood pressure.    -   15. The antibody or antibody fragment of any one of paragraphs 1        to 14, wherein said antibody or antibody fragment treats or        reduces the risk in said human of at least one condition        selected from a lipid disorder, hyperlipoproteinemia,        hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart        attack, a stroke, coronary heart disease, atherosclerosis,        peripheral vascular disease, claudication (eg, claudication        associated with elevated cholesterol) and high blood pressure.    -   16. The antibody or antibody fragment of any one of paragraphs 1        to 15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.    -   17. The antibody or antibody fragment of any one of paragraphs 1        to 16, wherein said antibody or antibody fragment is for        administration by intravenous or subcutaneous administration        and/or is comprised in an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining    previously reduced cholesterol level in a human in need thereof    or (b) targeting PCSK9 in a human, the method comprising    administering to said human an antibody or antibody fragment that    specifically binds a proprotein convertase subtilisin/kexin type 9    (PCSK9) that comprises a C-terminal domain comprising a said PCSK9    mutation (eg, I474V or E670G) in SEQ ID NO: 1, wherein the antibody    or fragment comprises a human gamma-1 heavy chain constant region    that comprises an Asp corresponding to position 204 of SEQ ID NO: 42    or a Leu corresponding to position 206 of SEQ ID NO: 42 and wherein    said human comprises (i) an IGHG1*01 human heavy chain constant    region gene segment, or the human expresses antibodies comprising    human gamma-1 heavy chain constant regions comprising such an Asp    and Leu and (ii) a nucleotide sequence encoding said proprotein    convertase subtilisin/kexin type 9 (PCSK9) that comprises a    C-terminal domain comprising said mutation (eg, I474V or E670G) in    SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation I474 in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-1 heavy        chain constant region that comprises an Asp corresponding to        position 204 of SEQ ID NO: 42 and a Leu corresponding to        position 206 of SEQ ID NO: 42 and wherein said human        comprises (i) an IGHG1*01 human heavy chain constant region gene        segment and (ii) a nucleotide sequence encoding said proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising said mutation I474V in SEQ ID        NO: 1. Optionally, the antibody or fragment comprises a IGHG1*01        human heavy chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   Another example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-1 heavy        chain constant region that comprises an Asp corresponding to        position 204 of SEQ ID NO: 42 and a Leu corresponding to        position 206 of SEQ ID NO: 42 and wherein said human        comprises (i) an IGHG1*01 human heavy chain constant region gene        segment and (ii) a nucleotide sequence encoding said proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising said mutation E670G in SEQ ID        NO: 1. Optionally, the antibody or fragment comprises an        IGHG1*01 human heavy chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        gamma-1 heavy chain constant region that comprises an Asp        corresponding to position 204 of SEQ ID NO: 42 and a Leu        corresponding to position 206 of SEQ ID NO: 42 and wherein said        human comprises (i) an IGHG1*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting a human comprising said nucleotide sequence of (ii),    wherein the human is the human of paragraph 1.-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human gamma-1 heavy chain constant region that comprises an Asp    corresponding to position 204 of SEQ ID NO: 42 and a Leu    corresponding to position 206 of SEQ ID NO: 42.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGHG1*01 human heavy chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation (eg,    I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474Vor E670G in    SEQ ID NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)    reducing cholesterol level or maintaining previously reduced    cholesterol level in a human in need thereof or (b) targeting PCSK9    in a human,    -   wherein the antibody comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro corresponding to position 72 of SEQ ID        NO: 44, an Asn corresponding to position 75 of SEQ ID NO: 44, a        Phe corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44, and the antibody        or fragment specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) amino acid sequence that        comprises a C-terminal domain comprising a said PCSK9 mutation        (eg, I474V or E670G) in SEQ ID NO:1, wherein the human comprises        a nucleotide sequence encoding said amino acid sequence and        comprises an IGHG2*01 human heavy chain constant region gene        segment, or the human expresses antibodies comprising human        gamma-2 constant regions comprising such a Pro, Asn, Phe, Val        and Ala.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody comprises a human gamma-2 heavy chain        constant region that comprises a Pro corresponding to position        72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ        ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,        a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44, and the antibody        or fragment specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) amino acid sequence that        comprises a C-terminal domain comprising a mutation I474V in SEQ        ID NO:1, wherein the human comprises a nucleotide sequence        encoding said amino acid sequence and comprises an IGHG2*01        human heavy chain constant region gene segment. Optionally, the        antibody or fragment comprises an IGHG2*01 human heavy chain        constant    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.region.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody comprises a human gamma-2 heavy chain        constant region that comprises a Pro corresponding to position        72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQ        ID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44,        a Val corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44, and the antibody        or fragment specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) amino acid sequence that        comprises a C-terminal domain comprising a mutation E670G in SEQ        ID NO:1, wherein the human comprises a nucleotide sequence        encoding said amino acid sequence and comprises an IGHG2*01        human heavy chain constant region gene segment. Optionally, the        antibody or fragment comprises an IGHG2*01 human heavy chain        constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        gamma-2 heavy chain constant region that comprises a Pro        corresponding to position 72 of SEQ ID NO: 44, an Asn        corresponding to position 75 of SEQ ID NO: 44, a Phe        corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44 and wherein said        human comprises (i) an IGHG2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    antibody comprises a human gamma-2 heavy chain constant region that    comprises a Pro corresponding to position 72 of SEQ ID NO: 44, an    Asn corresponding to position 75 of SEQ ID NO: 44, a Phe    corresponding to position 76 of SEQ ID NO: 44, a Val corresponding    to position 161 of SEQ ID NO: 44 and an Ala corresponding to    position 257 of SEQ ID NO: 44.-   3. The antibody or antibody fragment of paragraph 1 or 2, wherein    the antibody comprises an

IGHG2*01 human heavy chain constant region.

-   4. The antibody or antibody fragment of any one of paragraphs 1 to    3, wherein the human has been determined to comprise the nucleotide    sequence that encodes a PCSK9 comprising a C-terminal domain    comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or    a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30.-   5. The antibody or antibody fragment of any one of paragraphs 1 to    4, the method comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation (eg, I474V or E670G)    and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)    variant protein comprising said mutation (eg, I474V or E670G),    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   6. The antibody or antibody fragment of paragraph 5, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation (eg, I474V or E670G)    in SEQ ID NO: 1.-   7. The antibody or antibody fragment of paragraph 6, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   8. The antibody or antibody fragment of paragraph 6 or 7, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   9. The antibody or antibody fragment of any one of paragraphs 1 to    8, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 1 to    9, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   11. The antibody or antibody fragment of paragraph 9 or 10, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   12. The antibody or antibody fragment of any one of paragraphs 6 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a said    PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human    is further indicated as comprising the nucleotide sequence of SEQ ID    NO: 28, or said human is indicated as homozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G in SEQ ID NO: 1.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication (eg, claudication associated with    elevated cholesterol) and high blood pressure.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   16. The antibody or antibody fragment of any one of paragraphs 1 to    15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   17. The antibody or antibody fragment of any one of paragraphs 1 to    16, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining    previously reduced cholesterol level in a human in need thereof    or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a said PCSK9 mutation (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody or fragment        comprises a human gamma-2 heavy chain constant region that        comprises an amino acid selected from the group consisting of a        Pro corresponding to position 72 of SEQ ID NO: 44, an Asn        corresponding to position 75 of SEQ ID NO: 44, a Phe        corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44 and wherein said        human comprises (i) an IGHG2*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        such a Pro, Asn, Phe, Val and Ala and (ii) a nucleotide sequence        encoding said proprotein convertase subtilisin/kexin type 9        (PCSK9) that comprises a C-terminal domain comprising said        mutation (eg, I474V or E670G) in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation I474 in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-2 heavy        chain constant region that comprises a Pro corresponding to        position 72 of SEQ ID NO: 44, an Asn corresponding to position        75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ        ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44        and an Ala corresponding to position 257 of SEQ ID NO: 44 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment and (ii) a nucleotide sequence        encoding said proprotein convertase subtilisin/kexin type 9        (PCSK9) that comprises a C-terminal domain comprising said        mutation I474V in SEQ ID NO: 1. Optionally, the antibody or        fragment comprises an IGHG2*01 human heavy chain constant        region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   Another example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human gamma-2 heavy        chain constant region that comprises a Pro corresponding to        position 72 of SEQ ID NO: 44, an Asn corresponding to position        75 of SEQ ID NO: 44, a Phe corresponding to position 76 of SEQ        ID NO: 44, a Val corresponding to position 161 of SEQ ID NO: 44        and an Ala corresponding to position 257 of SEQ ID NO: 44 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment and (ii) a nucleotide sequence        encoding said proprotein convertase subtilisin/kexin type 9        (PCSK9) that comprises a C-terminal domain comprising said        mutation E670G in SEQ ID NO: 1. Optionally, the antibody or        fragment comprises an IGHG2*01 human heavy chain constant        region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        gamma-2 heavy chain constant region that comprises a Pro        corresponding to position 72 of SEQ ID NO: 44, an Asn        corresponding to position 75 of SEQ ID NO: 44, a Phe        corresponding to position 76 of SEQ ID NO: 44, a Val        corresponding to position 161 of SEQ ID NO: 44 and an Ala        corresponding to position 257 of SEQ ID NO: 44 and wherein said        human comprises (i) an IGHG2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human gamma-1 heavy chain constant region that comprises a Pro    corresponding to position 72 of SEQ ID NO: 44, an Asn corresponding    to position 75 of SEQ ID NO: 44, a Phe corresponding to position 76    of SEQ ID NO: 44, a Val corresponding to position 161 of SEQ ID NO:    44 and an Ala corresponding to position 257 of SEQ ID NO: 44.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGHG2*01 human heavy chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation (eg,    I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G in    SEQ ID NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence s SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-17 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)    reducing cholesterol level or maintaining previously reduced    cholesterol level in a human in need thereof or (b) targeting PCSK9    in a human,    -   wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 or a Cys corresponding to position        87 of SEQ ID NO: 50, and the antibody or fragment specifically        binds a proprotein convertase subtilisin/kexin type 9 (PCSK9)        amino acid sequence that comprises a C-terminal domain        comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID        NO:1, wherein the human comprises a nucleotide sequence encoding        said amino acid sequence and comprises an IGKC*01 human light        chain constant region gene segment, or the human expresses        antibodies comprising human kappa light chain constant regions        comprising such an Val and Cys.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50, and the antibody or fragment specifically        binds a proprotein convertase subtilisin/kexin type 9 (PCSK9)        amino acid sequence that comprises a C-terminal domain        comprising a mutation I474V in SEQ ID NO:1, wherein the human        comprises a nucleotide sequence encoding said amino acid        sequence and comprises an IGKC*01 human light chain constant        region gene segment. Optionally, the antibody or fragment        comprises an IGKC*01 human light chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50, and the antibody or fragment specifically        binds a proprotein convertase subtilisin/kexin type 9 (PCSK9)        amino acid sequence that comprises a C-terminal domain        comprising a mutation E670G in SEQ ID NO:1, wherein the human        comprises a nucleotide sequence encoding said amino acid        sequence and comprises an IGKC*01 human light chain constant        region gene segment. Optionally, the antibody or fragment        comprises an IGKC*01 human light chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        kappa light chain constant region that comprises a Val        corresponding to position 84 of SEQ ID NO: 50 and a Cys        corresponding to position 87 of SEQ ID NO: 50 and wherein said        human comprises (i) an IGKC*01 human heavy chain constant region        gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    antibody comprises a human kappa light chain constant region that    comprises a Val corresponding to position 84 of SEQ ID NO: 50 or a    Cys corresponding to position 87 of SEQ ID NO: 50.-   3. The antibody or antibody fragment of paragraph 1 or 2, wherein    the antibody comprises an IGKC*01 human kappa chain constant region.-   4. The antibody or antibody fragment of any one of paragraphs 1 to    3, wherein the human has been determined to comprise the nucleotide    sequence that encodes a PCSK9 comprising a C-terminal domain    comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or    a proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein encoded by the nucleotide sequence of SEQ ID NO: 29 or 30.-   5. The antibody or antibody fragment of any one of paragraphs 1 to    4, the method comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation (eg, I474V or E670G)    and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)    variant protein comprising said mutation (eg, I474V or E670G),    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   6. The antibody or antibody fragment of paragraph 5, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation (eg, I474V or E670G)    in SEQ ID NO: 1.-   7. The antibody or antibody fragment of paragraph 6, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   8. The antibody or antibody fragment of paragraph 6 or 7, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   9. The antibody or antibody fragment of any one of paragraphs 1 to    8, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 1 to    9, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   11. The antibody or antibody fragment of paragraph 9 or 10, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   12. The antibody or antibody fragment of any one of paragraphs 6 to    10, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a said    PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human    is further indicated as comprising the nucleotide sequence of SEQ ID    NO: 28, or said human is indicated as homozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain-   comprising a mutation I474V or E670G in SEQ ID NO: 1.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication (eg, claudication associated with    elevated cholesterol) and high blood pressure.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   16. The antibody or antibody fragment of any one of paragraphs 1 to    15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   17. The antibody or antibody fragment of any one of paragraphs 1 to    16, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-18 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining    previously reduced cholesterol level in a human in need thereof    or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a said PCSK9 mutation (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody or fragment        comprises a human kappa light chain constant region that        comprises a Val corresponding to position 84 of SEQ ID NO: 50 or        a Cys corresponding to position 87 of SEQ ID NO: 50 and wherein        said human comprises (i) an IGKC*01 human light chain constant        region gene segment, or the human expresses antibodies        comprising human kappa light chain constant regions comprising        such an Val and Cys and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation I474 in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50 and wherein said human comprises (i) an        IGKC*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   Another example provides:—    -   A method of (a) reducing cholesterol level or maintaining        previously reduced cholesterol level in a human in need thereof        or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a mutation E670G in SEQ ID NO: 1,        wherein the antibody or fragment comprises a human kappa light        chain constant region that comprises a Val corresponding to        position 84 of SEQ ID NO: 50 and a Cys corresponding to position        87 of SEQ ID NO: 50 and wherein said human comprises (i) an        IGKC*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation E670G in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGKC*01 human        light chain constant region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        kappa light chain constant region that comprises a Val        corresponding to position 84 of SEQ ID NO: 50 and a Cys        corresponding to position 87 of SEQ ID NO: 50 and wherein said        human comprises (i) an IGKC*01 human heavy chain constant region        gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the antibody comprises a    human kappa light chain constant region that comprises a Val    corresponding to position 84 of SEQ ID NO: 50 or a Cys corresponding    to position 87 of SEQ ID NO: 50.-   4. The method of paragraph 1, 2 or 3, wherein the antibody comprises    an IGKC*01 human kappa chain constant region.-   5. The method of any one of paragraphs 1 to 4, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation (eg,    I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   6. The method of any one of paragraphs 1 to 5, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   7. The method of paragraph 6, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   8. The method of paragraph 7, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   9. The method of paragraph 7 or 8, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   10. The method of any one of paragraphs 1 to 9, wherein said human    is or has been further determined to be substantially resistant to    statin treatment.-   11. The method of any one of paragraphs 1 to 10, wherein said human    is receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   12. The method of paragraph 10 or 11, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   13. The method of any one of paragraphs 7 to 9, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   14. The method of any one of paragraphs 1 to 13, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G in    SEQ ID NO: 1.-   15. The method of any one of paragraphs 1 to 14, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   17. The method of any one of paragraphs 1 to 16, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   18. The method of any one of paragraphs 1 to 17, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)    reducing cholesterol level or maintaining previously reduced    cholesterol level in a human in need thereof or (b) targeting PCSK9    in a human,    -   wherein the antibody or fragment comprises a human IGLC2*01        lambda light chain constant region, and the antibody or fragment        specifically binds a proprotein convertase subtilisin/kexin type        9 (PCSK9) amino acid sequence that comprises a C-terminal domain        comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID        NO:1, wherein the human comprises a nucleotide sequence encoding        said amino acid sequence and comprises a human IGLC2*01 lambda        light chain constant region gene segment, or the human expresses        antibodies comprising human IGLC2*01 lambda light chain constant        regions.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   An example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody or fragment comprises a human IGLC2*01        lambda light chain constant region, and the antibody or fragment        specifically binds a proprotein convertase subtilisin/kexin type        9 (PCSK9) amino acid sequence that comprises a C-terminal domain        comprising a mutation I474V in SEQ ID NO:1, wherein the human        comprises a nucleotide sequence encoding said amino acid        sequence and comprises a human IGLC2*01 lambda light chain        constant region gene segment. Optionally, the antibody or        fragment comprises a human IGLC2*01 lambda light chain constant        region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   Another example provides:—    -   An antibody or antibody fragment for use in a method of (a)        reducing cholesterol level or maintaining previously reduced        cholesterol level in a human in need thereof or (b) targeting        PCSK9 in a human,    -   wherein the antibody or fragment comprises a human IGLC2*01        lambda light chain constant region, and the antibody or fragment        specifically binds a proprotein convertase subtilisin/kexin type        9 (PCSK9) amino acid sequence that comprises a C-terminal domain        comprising a mutation E670G in SEQ ID NO:1, wherein the human        comprises a nucleotide sequence encoding said amino acid        sequence and comprises a human IGLC2*01 lambda light chain        constant region gene segment. Optionally, the antibody or        fragment comprises a human IGLC2*01 lambda light chain constant        region.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        IGLC2*01 lambda light chain constant region and wherein said        human comprises (i) an IGLC2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   2. The antibody or antibody fragment of paragraph 1, wherein the    human has been determined to comprise the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded    by the nucleotide sequence of SEQ ID NO: 29 or 30.-   3. The antibody or antibody fragment of paragraphs 1 or 2, the    method comprising the step of determining that the human comprises    the nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation (eg, I474V or E670G) and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein comprising said mutation (eg, I474V or E670G), optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   4. The antibody or antibody fragment of paragraph 3, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation (eg, I474V or E670G)    in SEQ ID NO: 1.-   5. The antibody or antibody fragment of paragraph 4, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   6. The antibody or antibody fragment of paragraph 4 or 5, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   7. The antibody or antibody fragment of any one of paragraphs 1 to    6, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   8. The antibody or antibody fragment of any one of paragraphs 1 to    7, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   9. The antibody or antibody fragment of paragraph 7 or 8, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 4 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   11. The antibody or antibody fragment of any one of paragraphs 1 to    10, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a said    PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human    is further indicated as comprising the nucleotide sequence of SEQ ID    NO: 28, or said human is indicated as homozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G in SEQ ID NO: 1.-   12. The antibody or antibody fragment of any one of paragraphs 1 to    11, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication (eg, claudication associated with    elevated cholesterol) and high blood pressure.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-16 as follows.

-   1. A method of reducing cholesterol level or maintaining previously    reduced cholesterol level in a human in need thereof, the method    comprising administering to said human an antibody or antibody    fragment that specifically binds a proprotein convertase    subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal domain    comprising a said PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO:    1, wherein the antibody or fragment comprises a human IGLC2*01    lambda light chain constant region and wherein said human    comprises (i) an IGLC2*01 human light chain constant region gene    segment, or the human expresses antibodies comprising a human    IGLC2*01 lambda light chain constant regions and (ii) a nucleotide    sequence encoding said proprotein convertase subtilisin/kexin type 9    (PCSK9) that comprises a C-terminal domain comprising said mutation    (eg, I474V or E670G) in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   An example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation I474 in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human IGLC2*01 lambda light        chain constant region and wherein said human comprises (i) an        IGLC2*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation I474V in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGLC2*01 human        light chain constant region.    -   Another example provides:—    -   A method of reducing cholesterol level or maintaining previously        reduced cholesterol level in a human in need thereof, the method        comprising administering to said human an antibody or antibody        fragment that specifically binds a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a mutation E670G in SEQ ID NO: 1, wherein the        antibody or fragment comprises a human IGLC2*01 lambda light        chain constant region and wherein said human comprises (i) an        IGLC2*01 human light chain constant region gene segment and (ii)        a nucleotide sequence encoding said proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising said mutation E670G in SEQ ID NO: 1.        Optionally, the antibody or fragment comprises an IGLC2*01 human        light chain constant region.    -   In an example, said antibody or antibody fragment has been        determined to specifically bind a proprotein convertase        subtilisin/kexin type 9 (PCSK9) that comprises a C-terminal        domain comprising a said PCSK9 mutation (eg, I474V or E670G) in        SEQ ID NO: 1, wherein the antibody or fragment comprises a human        IGLC2*01 lambda light chain constant region and wherein said        human comprises (i) an IGLC2*01 human heavy chain constant        region gene segment and (ii) a nucleotide sequence encoding said        proprotein convertase subtilisin/kexin type 9 (PCSK9) that        comprises a C-terminal domain comprising said mutation (eg,        I474V or E670G) in SEQ ID NO: 1-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the human has been    determined to comprise the nucleotide sequence that encodes a PCSK9    comprising a C-terminal domain comprising said mutation (eg, I474V    or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   4. The method of any one of paragraphs 1 to 3, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   5. The method of paragraph 4, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   6. The method of paragraph 5, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   7. The method of paragraph 5 or 6, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   8. The method of any one of paragraphs 1 to 7, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   9. The method of any one of paragraphs 1 to 8, wherein said human is    receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   10. The method of paragraph 8 or 9, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   11. The method of any one of paragraphs 5 to 7, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   12. The method of any one of paragraphs 1 to 11, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G in    SEQ ID NO: 1.-   13. The method of any one of paragraphs 1 to 12, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   14. The method of any one of paragraphs 1 to 13, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   15. The method of any one of paragraphs 1 to 14, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

Further exemplary ligands are in the paragraphs 1-15 as follows.

-   1. An antibody or antibody fragment for use in a method of (a)    reducing cholesterol level or maintaining previously reduced    cholesterol level in a human in need thereof or (b) targeting PCSK9    in a human, wherein the antibody or fragment comprises a human    variable domain that is derived from the recombination of a human VH    gene segment, a human D gene segment and a human JH gene segment,    wherein the VH gene segment is as defined in any one of clauses 80    to 90, and the antibody or fragment specifically binds a proprotein    convertase subtilisin/kexin type 9 (PCSK9) amino acid sequence that    comprises a C-terminal domain comprising a said PCSK9 mutation (eg,    I474V or E670G) in SEQ ID NO:1, wherein the human comprises a    nucleotide sequence encoding said amino acid sequence, and comprises    said VH gene segment or expresses antibodies comprising VH domains    that are derived from the recombination of said human VH gene    segment, a human D gene segment and a human JH gene segment.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.-   2. The antibody or antibody fragment of paragraph 1, wherein the    human has been determined to comprise the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) in SEQ ID NO: 1 and/or a proprotein    convertase subtilisin/kexin type 9 (PCSK9) variant protein encoded    by the nucleotide sequence of SEQ ID NO: 29 or 30.-   3. The antibody or antibody fragment of paragraphs 1 or 2, the    method comprising the step of determining that the human comprises    the nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation (eg, I474V or E670G) and/or a    proprotein convertase subtilisin/kexin type 9 (PCSK9) variant    protein comprising said mutation (eg, I474V or E670G), optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   4. The antibody or antibody fragment of paragraph 3, wherein the    step of determining comprises assaying a biological sample from the    human for a nucleotide sequence encoding the PCSK9 that comprises    the C-terminal domain comprising the mutation (eg, I474V or E670G)    in SEQ ID NO: 1.-   5. The antibody or antibody fragment of paragraph 4, wherein the    assaying comprises contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   6. The antibody or antibody fragment of paragraph 4 or 5, wherein    the assaying comprises nucleic acid amplification and optionally one    or more methods selected from sequencing, next generation    sequencing, nucleic acid hybridization, and allele-specific    amplification and/or wherein the assaying is performed in a    multiplex format.-   7. The antibody or antibody fragment of any one of paragraphs 1 to    6, wherein said antibody or antibody fragment is for administration    to a human that is or has been further determined to be    substantially resistant to statin treatment.-   8. The antibody or antibody fragment of any one of paragraphs 1 to    7, wherein antibody or antibody fragment is for administration to a    human that is receiving or has received statin treatment or has    reduced responsiveness to statin treatment.-   9. The antibody or antibody fragment of paragraph 7 or 8, wherein    said antibody or antibody fragment is for administration to the    human separately or simultaneously with said statin treatment.-   10. The antibody or antibody fragment of any one of paragraphs 4 to    8, wherein said biological sample comprises serum, blood, faeces,    tissue, a cell, urine and/or saliva of said human.-   11. The antibody or antibody fragment of any one of paragraphs 1 to    10, wherein said human is indicated as heterozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a said    PCSK9 mutation (eg, I474V or E670G), optionally, wherein said human    is further indicated as comprising the nucleotide sequence of SEQ ID    NO: 28, or said human is indicated as homozygous for a nucleotide    sequence encoding the PCSK9 C-terminal domain comprising a mutation    I474V or E670G in SEQ ID NO: 1.-   12. The antibody or antibody fragment of any one of paragraphs 1 to    11, wherein said human has been diagnosed with at least one    condition selected from a lipid disorder, hyperlipoproteinemia,    hyperlipidemia, dyslipidemia, hypercholesterolemia, a heart attack,    a stroke, coronary heart disease, atherosclerosis, peripheral    vascular disease, claudication (eg, claudication associated with    elevated cholesterol) and high blood pressure.-   13. The antibody or antibody fragment of any one of paragraphs 1 to    12, wherein said antibody or antibody fragment treats or reduces the    risk in said human of at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   14. The antibody or antibody fragment of any one of paragraphs 1 to    13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.-   15. The antibody or antibody fragment of any one of paragraphs 1 to    14, wherein said antibody or antibody fragment is for administration    by intravenous or subcutaneous administration and/or is comprised in    an injectable preparation.

Further exemplary methods are in the paragraphs 1-16 as follows.

-   1. A method of (a) reducing cholesterol level or maintaining    previously reduced cholesterol level in a human in need thereof    or (b) targeting PCSK9 in a human,    -   the method comprising administering to said human an antibody or        antibody fragment that specifically binds a proprotein        convertase subtilisin/kexin type 9 (PCSK9) that comprises a        C-terminal domain comprising a said PCSK9 mutation (eg, I474V or        E670G) in SEQ ID NO: 1, wherein the antibody or fragment        comprises a human variable domain that is derived from the        recombination of a human VH gene segment, a human D gene segment        and a human JH gene segment, wherein the VH gene segment is as        defined in any one of clauses 80 to 90 and wherein said human        comprises (i) comprises said VH gene segment or expresses        antibodies comprising VH domains that are derived from the        recombination of said human VH gene segment, a human D gene        segment and a human JH gene segment and (ii) a nucleotide        sequence encoding said proprotein convertase subtilisin/kexin        type 9 (PCSK9) that comprises a C-terminal domain comprising        said mutation (eg, I474V or E670G) in SEQ ID NO: 1.    -   In an embodiment, said mutation is I474V. In another embodiment,        said mutation is E670G.    -   In an embodiment of (b), the antibody or fragment treats or        reduces cholesterol level or maintains previously reduced        cholesterol level in the human.-   2. The method of paragraph 1, comprising, before said administering,    selecting said human comprising said nucleotide sequence of (ii).-   3. The method of paragraph 1 or 2, wherein the human has been    determined to comprise the nucleotide sequence that encodes a PCSK9    comprising a C-terminal domain comprising said mutation (eg, I474V    or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   4. The method of any one of paragraphs 1 to 3, comprising the step    of determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   5. The method of paragraph 4, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   6. The method of paragraph 5, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   7. The method of paragraph 5 or 6, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   8. The method of any one of paragraphs 1 to 7, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   9. The method of any one of paragraphs 1 to 8, wherein said human is    receiving or has received statin treatment or has reduced    responsiveness to statin treatment.-   10. The method of paragraph 8 or 9, wherein said antibody or    antibody fragment is administered to the human separately or    simultaneously with said statin treatment.-   11. The method of any one of paragraphs 5 to 7, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   12. The method of any one of paragraphs 1 to 11, wherein said human    is indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474Vor E670G in    SEQ ID NO: 1.-   13. The method of any one of paragraphs 1 to 12, wherein said human    has been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   14. The method of any one of paragraphs 1 to 13, wherein said    antibody or antibody fragment treats or reduces the risk in said    human of at least one condition selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   15. The method of any one of paragraphs 1 to 14, wherein the    nucleotide sequence is SEQ ID NO: 29 or 30.-   16. The method of any one of paragraphs 1 to 15, wherein said    antibody or antibody fragment is administered by intravenous or    subcutaneous administration and/or is comprised in an injectable    preparation.

In an alternative, instead of being a method “of reducing cholesterollevel or maintaining previously reduced cholesterol level” as rectiedherein the method is “of treating and/or preventing a humanPCSK9-mediated condition or disease” (eg, associated with hyper- orhypocholesterolemia or any other PCSK9-mediated disease or conditiondisclosed herein) and the mutation is instead selected from the group ofR46L, A53V, N425S, A443T, I474V, Q619P and E670G. Hence in thisalternative, any such embodiment is disclosed as written herein but withthe mutation recited as selected from this group. In an example, themutation is R46L. In an example, the mutation is A53V. In an example,the mutation is N425S. In an example, the mutation is A443T. In anexample, the mutation is I474V. In an example, the mutation is Q619P. Inan example, the mutation is E670G.

Regimens

A: The invention further provides the following regimens, ligands andkits.

-   1. A method for treating a human PCSK9-mediated disease or condition    in a human by targeting a rare variant human PCSK9, the method    comprising administering to the human a ligand (eg, an antibody or    fragment) that has been determined to specifically bind to said    PCSK9variant; wherein the human expresses said PCSK9 variant or the    genome of the human comprises a nucleotide sequence encoding said    PCSK9 variant; wherein said human is treated for said disease or    condition.    -   In an alternative, clause 1 provides:—    -   A method for targeting a rare variant human PCSK9, the method        comprising administering to the human a ligand (eg, an antibody        or fragment) that has been determined to specifically bind to        said PCSK9variant; wherein the human expresses said PCSK9        variant or the genome of the human comprises a nucleotide        sequence encoding said PCSK9 variant. In an embodiment, said        human is treated for said disease or condition.        The variant PCSK9 can, for example, be any rare variant as        described herein.        For example, there is provided:        1a. A method for treating a PCSK9-mediated disease or condition        in a human by targeting a PCSK9 that comprises a C-terminal        domain amino acid polymorphism (compared to SEQ ID NO: 1), the        method comprising administering to the human a ligand (eg, an        antibody or fragment) that has been determined to specifically        bind to a PCSK9 comprising a C-terminal domain comprising a said        PCSK9 mutation (eg, I474V or 670G) (numbering according to SEQ        ID NO:1); wherein the human expresses said PCSK9 or the genome        of the human comprises a nucleotide sequence encoding said        PCSK9; wherein said human is treated for said disease or        condition.        For example, there is provided:        1b. A method for targeting a PCSK9 that comprises a C-terminal        domain amino acid polymorphism (compared to SEQ ID NO: 1), the        method comprising administering to the human a ligand (eg, an        antibody or fragment) that has been determined to specifically        bind to a PCSK9 comprising a C-terminal domain comprising a said        PCSK9 mutation (eg, I474V or 670G) (numbering according to SEQ        ID NO:1); wherein the human expresses said PCSK9 or the genome        of the human comprises a nucleotide sequence encoding said        PCSK9; optionally wherein said human is treated for said disease        or condition.        In an embodiment, determination of said specific binding is by        reference to binding assay data, eg, as determined using SPR or        ELISA. Determination may, for example, be by reference to        information in a printed publication, eg, with knowledge of data        presented in the present or another patent application or in a        journal article. Once armed with such knowledge (eg, in the        absence of further testing of binding), the skilled person is        able—by direction of the present invention—to treat a relevant        human whose genotype or phenotype matches the binding        specificity of the ligand.        The antibody or fragment can be according to any configuration,        example, embodiment, aspect, clause or paragraph herein.        In an embodiment, the method comprises, before said        administering, selecting a human comprising said nucleotide        sequence encoding the PCSK9, wherein the human is said human in        clause 1 (eg, 1a).-   2. The method of clause 1, comprising before said administering the    step of determining that the ligand specifically binds to said    PCSK9, eg, using SPR or ELISA.-   3. The method of clause 1 or 2, wherein the specific binding to said    PCSK9 is binding with a dissociation constant (Kd) of 1 nM or less,    eg, 100, 10 or 1 pM or less.-   4. The method of any of clauses 1 to 3 (eg, clause 1a), wherein the    condition is elevated LDL-cholesterol or is caused by elevated    LDL-cholesterol.-   5. The method of clause 4 (eg, when dependent from clause 1a),    wherein the condition is selected from a lipid disorder,    hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   6. The method of any one of clauses 1 to 5 (eg, when dependent from    clause 1a), wherein the human has been determined to comprise the    nucleotide sequence that encodes a PCSK9 comprising a C-terminal    domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1    and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)    variant protein encoded by the nucleotide sequence of SEQ ID NO: 29    or 30.-   7. The method of any one of clauses 1 to 6 (eg, when dependent from    clause 1a), comprising the step of determining that the human    comprises the nucleotide sequence that encodes a PCSK9 comprising a    C-terminal domain comprising said mutation (eg, I474V or E670G)    and/or a proprotein convertase subtilisin/kexin type 9 (PCSK9)    variant protein comprising said mutation (eg, I474V or E670G),    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   8. The method of clause 7, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding the PCSK9 that comprises the C-terminal domain    comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   9. The method of clause 8, wherein the assaying comprises contacting    the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.    -   10. The method of clause 8 or 9, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   11. The method of any one of clauses 1 to 10, wherein said human        is or has been further determined to be substantially resistant        to statin treatment.    -   12. The method of any one of clauses 1 to 11, wherein said human        is receiving or has received statin treatment or has reduced        responsiveness to statin treatment.    -   13. The method of clauses 11 or 12, wherein said antibody or        antibody fragment is administered to the human separately or        simultaneously with said statin treatment.    -   14. The method of any one of clauses 8 to 10, wherein said        biological sample comprises serum, blood, faeces, tissue, a        cell, urine and/or saliva of said human.    -   15. The method of any one of clauses 1 to 14, wherein said human        is indicated as heterozygous for a nucleotide sequence encoding        the PCSK9 C-terminal domain comprising said mutation (eg, I474V        or E670G), optionally, wherein said human is further indicated        as comprising the nucleotide sequence of SEQ ID NO: 28, or said        human is indicated as homozygous for a nucleotide sequence        encoding the PCSK9 C-terminal domain comprising said mutation        I474V or E670G in SEQ ID NO: 1.    -   16. The method of any one of clauses 1 to 15, wherein said human        has been diagnosed with at least one condition selected from a        lipid disorder, hyperlipoproteinemia, hyperlipidemia,        dyslipidemia, hypercholesterolemia, a heart attack, a stroke,        coronary heart disease, atherosclerosis, peripheral vascular        disease, claudication (eg, claudication associated with elevated        cholesterol) and high blood pressure.    -   17. The method of any one of clauses 1 to 16, wherein said        antibody or antibody fragment treats or reduces the risk in said        human of at least one condition selected from a lipid disorder,        hyperlipoproteinemia, hyperlipidemia, dyslipidemia,        hypercholesterolemia, a heart attack, a stroke, coronary heart        disease, atherosclerosis, peripheral vascular disease,        claudication (eg, claudication associated with elevated        cholesterol) and high blood pressure.    -   18. The method of any one of clauses 1 to 17, wherein the        nucleotide sequence is SEQ ID NO: 29 or 30.    -   19. The method of any one of clauses 1 to 18, wherein said        antibody or antibody fragment is administered by intravenous or        subcutaneous administration and/or is comprised in an injectable        preparation.    -   20. A ligand (eg, an antibody or fragment) for use in the method        of any one of clauses 1 to 19, wherein the ligand specifically        binds the PCSK9.    -   21. A kit comprising the ligand of clause 20 and instructions        for carrying out the method of any one of clauses 1 to 19.

B: The invention further provides the following regimens, ligands andkits.

-   1. A method of reducing cholesterol level or maintaining a    previously reduced cholesterol level in a human in need thereof, the    method comprising:—    -   a. Carrying out an initial treatment of said human for an        initial treatment period by administering an anti-human PCSK9        ligand (eg, an antibody or fragment) to said human, wherein (i)        the ligand has been determined to specifically bind to a PCSK9        comprising a C-terminal domain comprising a said PCSK9 mutation        (eg, I474V or 670G) (numbering according to SEQ ID NO:1); (ii)        the human expresses said PCSK9 or the genome of the human        comprises a nucleotide sequence encoding said PCSK9 and (iii)        the human has received or is receiving statin treatment to lower        or maintain cholesterol level; wherein the initial treatment        comprises the administration of a single or multiple doses of        the ligand to the human;    -   b. Determining to (i) terminate statin treatment (ii) keep the        human off statin treatment; or (iii) reduce statin treatment        after said initial treatment period; and    -   c. Continuing to administer the ligand to said patient after        said time period has expired, thereby reducing cholesterol level        or maintaining a previously reduced cholesterol level in said        human.        In an embodiment, determination of said specific binding is by        reference to binding assay data, eg, as determined using SPR or        ELISA. Determination may, for example, be by reference to        information in a printed publication, eg, with knowledge of data        presented in the present or another patent application or in a        journal article. Once armed with such knowledge (eg, in the        absence of further testing of binding), the skilled person is        able—by direction of the present invention—to treat a relevant        human whose genotype or phenotype matches the binding        specificity of the ligand.        The antibody or fragment can be according to any configuration,        example, embodiment, aspect, clause or paragraph herein.        A pharmaceutically-effective amount of said ligand is        administered.        In an embodiment, the method comprises, before said        administering, selecting a human comprising said nucleotide        sequence encoding the PCSK9, wherein the human is said human        recited in clause 1.        In an example, the initial treatment period is 7 days, 14 days,        21 days, 28 days, a month, two months, three months, four        months, five months, six months, seven months, eight months,        nine months or a year.-   2. The method of clause 1, wherein the human has or is suffering    from statin-associated memory loss or a statin-associated    neurodegenerative condition, or has or is at increased risk of    diabetes (eg, statin-associated diabetes).-   3. The method of clause 1 or 2, comprising, before said initial    treatment, the step of determining that the human has or is    suffering from statin-associated memory loss or a statin-associated    neurodegenerative condition, or has or is at increased risk of    diabetes (eg, statin-associated diabetes).-   4. The method of clause 2 or 3, comprising, after step (b) (eg,    during step (c)) determining that the memory loss or said    neurodegenerative condition has improved.-   5. The method of any one of clauses 1 to 4, wherein said human is    over 40 years of age (eg, 50 or over, 55 or over, 60 or over, 65 or    over, or 70 or over).-   6. The method of any one of clauses 1 to 5, wherein step (c)    comprises determining to increase the doses of said ligand to be    administered after said initial treatment period and administering    said increased doses to said human.-   7. The method of any one of clauses 1 to 6, wherein step (b)    comprises determining that the human is intolerant or refactory to    treatment by a statin.-   8. The method of any one of clauses 1 to 7, wherein the initial    treatment comprises the administration of a statin, fenofibrate (eg,    Tricor™ or Lofibra™) or ezetimibe to the human in addition to the    ligand.-   9. The method of any one of clauses 1 to 8, wherein step (b)    comprises terminating or reducing statin, fenofibrate (eg, Tricor™    or Lofibra™) or ezetimibe treatment during step (c).-   10. The method of any one of clauses 1 to 9, comprising increasing    (ie, increasing compared to the initial treatment dose) the ligand    dose during step (c).-   11. The method of any one of clauses 1 to 10, wherein the human has    received high dose statin treatment prior to the initial treatment,    and wherein step (c) comprises administering a lower (eg, a medium    or low) dose statin treatment in addition to said ligand.    -   The skilled person is familiar with the meaning of high, medium        and low dose treatments (and how to determine according to each        patient, eg, the patient's body mass). For example, the statin        is selected from rosuvastatin, atorvastatin and simvastatin.    -   For example daily statin doses are as follows:—Low 10 to 20 mg        (eg, 10 mg); medium >20 and <60 mg (eg, 40 mg); high 60-100 mg        (eg, 80 mg).-   12. The method of any one of clauses 1 to 10, wherein the human has    received medium dose statin treatment prior to the initial    treatment, and wherein step (c) comprises administering a lower (eg,    a low) dose statin treatment or no statin in addition to said    ligand.-   13. The method of any one of clauses 1 to 10, wherein the human has    received low dose statin treatment prior to the initial treatment,    and wherein step (c) comprises administering no statin in addition    to said ligand.-   14. The method of any one of clauses 1 to 13, comprising, before the    initial treatment, the step of determining that the ligand    specifically binds to said PCSK9, eg, using SPR or ELISA.-   15. The method of any one of clauses 1 to 14, wherein the specific    binding to said PCSK9 is binding with a dissociation constant (Kd)    of 1 nM or less, eg, 100, 10 or 1 pM or less.-   16. The method of any of clauses 1 to 15, wherein the human is at    risk of or suffering from a condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   17. The method of clause 16, wherein step (c) treats or reduces the    risk of said condition in the human.-   18. The method of any one of clauses 1 to 17, wherein the human has    been determined to comprise the nucleotide sequence that encodes a    PCSK9 comprising a C-terminal domain comprising said mutation (eg,    I474V or E670G) in SEQ ID NO: 1 and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein encoded by the    nucleotide sequence of SEQ ID NO: 29 or 30.-   19. The method of any one of clauses 1 to 18, comprising the step of    determining that the human comprises the nucleotide sequence that    encodes a PCSK9 comprising a C-terminal domain comprising said    mutation (eg, I474V or E670G) and/or a proprotein convertase    subtilisin/kexin type 9 (PCSK9) variant protein comprising said    mutation (eg, I474V or E670G), optionally, wherein the determining    step is performed before administration of the antibody to the    human.-   20. The method of clause 19, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding the PCSK9 that comprises the C-terminal    domain comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1.-   21. The method of clause 20, wherein the assaying comprises    contacting the biological sample with    -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to at least one nucleotide        present in said selected sequence which is not present in SEQ ID        NO: 28 or hybridizes to an antisense sequence thereby forming a        complex when at least one nucleotide sequence encoding the PCSK9        that comprises the C-terminal domain comprising the mutation        (eg, I474V or E670G) in SEQ ID NO: 1 is present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        encoding the PCSK9 that comprises the C-terminal domain        comprising the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises at        least one nucleotide present in said selected sequence which is        not present in SEQ ID NO: 28 thereby forming a complex when the        nucleotide sequence encoding the PCSK9 that comprises a        C-terminal domain comprising the mutation (eg, I474V or E670G)        in SEQ ID NO: 1 is present; and detecting the presence or        absence of the complex, wherein detecting the presence of the        complex determines that the human comprises the PCSK9 that        comprises the C-terminal domain comprising the mutation (eg,        I474V or E670G) in SEQ ID NO: 1.-   22. The method of clause 20 or 21, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   23. The method of any one of clauses 1 to 22, wherein said human is    or has been further determined to be substantially resistant to    statin treatment.-   24. The method of any one of clauses 20 to 22, wherein said    biological sample comprises serum, blood, faeces, tissue, a cell,    urine and/or saliva of said human.-   25. The method of any one of clauses 1 to 24, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation (eg, I474V or    E670G), optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 28, or said human    is indicated as homozygous for a nucleotide sequence encoding the    PCSK9 C-terminal domain comprising said mutation I474V or E670G in    SEQ ID NO: 1.-   26. The method of any one of clauses 1 to 25, wherein said human has    been diagnosed with at least one condition selected from a lipid    disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia,    hypercholesterolemia, a heart attack, a stroke, coronary heart    disease, atherosclerosis, peripheral vascular disease, claudication    (eg, claudication associated with elevated cholesterol) and high    blood pressure.-   27. The method of any one of clauses 1 to 26, wherein the nucleotide    sequence is SEQ ID NO: 29 or 30.-   28. The method of any one of clauses 1 to 27, wherein said ligand    (eg, antibody or antibody fragment) is administered by intravenous    or subcutaneous administration and/or is comprised in an injectable    preparation.-   29. A ligand (eg, an antibody or fragment) for use in the method of    any one of clauses 1 to 28, wherein the ligand specifically binds    the PCSK9.-   30. A kit comprising the ligand of clause 29 and instructions for    carrying out the method of any one of clauses 1 to 28.

In an example of any aspect of the invention, the ligand (eg, antibodyor fragment, eg, alirocumab, bocovizumab or evolocumab, or an antibodyor fragment comprising the variable domains of 316P or 31H4) isadministered to the human at a two-weekly dose of from 75 to 150 mg (eg,from 75 to 150 mg administered once or twice over a two-week period). Inan example, the ligand is for such administration to the human.

Determination of Specific Binding of Ligands of the Invention to PCSK9Variants

Method of SPR Determination of Binding

Binding of the antibodies to the PCSK9 variants was carried out by SPRusing the ProteOn XPR36™ Array system (BioRad). An anti-human IgGsurface (Jackson Labs 109-005-008) was created on a GLC Biosensor chipby primary amine coupling. Test antibodies were captured on this surfaceas ligands. The PCSK9 variants were used as analytes and passed over thecaptured antibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Bindingcurves were double referenced using a buffer injection (i.e. 0 nM) toremove baseline drift and injection artefacts. Regeneration of thecapture surface was with 100 mM phosphoric acid which removed thecaptured antibody allowing another cycle of capture and binding. Thebinding sensorgrams generated were analysed using the 1:1 model inherentto the ProteOn XPR36 Array system analysis software. The assay wasperformed at 25° C. and using 1xHBS-EP (Teknova) as running buffer.

Data

Three antibodies were tested and the resulting binding data arepresented below (Table 3). Antibodies 316P and 31H4 are antibodiesdisclosed in US20110065902A1 (the sequences of these antibodies andtheir variable domains are incorporated herein by reference for possibleuse in the present invention and possible inclusion in claims herein).Antibody 316P comprises heavy chain variable domains derived fromrecombination of human VH3-23*04 and JH2*01 (with a D), and light chainvariable domains derived from recombination of human Vκ4-1*01 andJκ2*01.

Evolocumab comprises a human IGHG2*01 heavy chain and a human IGLC2*01lambda light chain, a VH derived from recombination of human IGHV1-18*01and IGHJ6*01 (with a D segment) and a Vλ derived from recombination ofhuman IGLV2-14*01 and IGLJ2*01.

TABLE 3 SPR Determination of Ligand Binding Specificity for PCSK9Variants Variant/Antibody ka (1/Ms) kd (1/s) KD (nM) PCSK9 a 316P1.37E+06 2.75E−04 0.201 31H4 1.14E+06 6.38E−05 0.056 Evolocumab 1.14E+052.62E−05 0.229 PCSK9 a′ 316P 1.50E+06 2.72E−04 0.181 31H4 1.23E+066.06E−05 0.049 Evolocumab 1.24E+05 2.29E−05 0.185 PCSK9 c 316P 1.49E+062.75E−04 0.184 31H4 1.22E+06 5.69E−05 0.047 Evolocumab 1.20E+05 2.20E−050.183 PCSK9 r 316P 1.40E+06 2.76E−04 0.197 31H4 1.15E+06 5.82E−05 0.051Evolocumab 1.16E+05 2.67E−05 0.230 PCSK9 f 316P 1.39E+06 2.82E−04 0.20331H4 1.13E+06 5.95E−05 0.053 Evolocumab 1.16E+05 2.66E−05 0.229 PCSK9 p316P 1.39E+06 2.73E−04 0.196 31H4 1.14E+06 6.12E−05 0.054 Evolocumab1.14E+05 2.50E−05 0.219

Results

The results showed that all antibodies tested bound to PCSK9 variantsequally, with any binding variation seen being within experimental errorfor such a strong affinity interaction.

Thus, the invention determines that an antibody with the followingprofile can specifically bind one or more variants of the invention:—

1. An antibody (eg, 316P or alirocumab) that comprises heavy chainvariable domains derived from recombination of human IGHV3-23*04 andIGHJH2*01 (with a D), and light chain variable domains derived fromrecombination of human IGKV4-1*01 and IGKJ2*01; or

2. An antibody (eg, evolocumab) that comprises human heavy chainvariable domains derived from recombination of human IGHV1-18*01 andIGHJ6*01 (with a D segment) and light chain variable domains derivedfrom recombination of human IGLV2-14*01 and IGLJ2*01.

Thus, according to the invention, the skilled person is hereby providedwith the required determination of specific binding of the ligand toPCSK9 variants. Applications of this determination are set out above inthe context of methods and other aspects of the invention.

References

-   Horton et al, Trends Biochem Sci. 2007 February; 32(2):71-7. Epub    2007 Jan. 9, Molecular biology of PCSK9: its role in LDL metabolism.-   Seidah and Prat, J Mol Med (Berl). 2007 July; 85(7):685-96. Epub    2007 Mar. 10, The proprotein convertases are potential targets in    the treatment of dyslipidemia.-   Benjannet et al, J Biol Chem. 2004 Nov. 19; 279(47):48865-75. Epub    2004 Sep. 9, NARC-1/PCSK9 and its natural mutants: zymogen cleavage    and effects on the low density lipoprotein (LDL) receptor and LDL    cholesterol.-   Lagace et al, J Clin Invest. 2006 November; 116(11):2995-3005,    Secreted PCSK9 decreases the number of LDL receptors in hepatocytes    and in livers of parabiotic mice.-   Maxwell et al, Proc Natl Acad Sci USA. 2005 Feb. 8; 102(6):2069-74.    Epub 2005 Jan. 27,-   Overexpression of PCSK9 accelerates the degradation of the LDLR in a    post-endoplasmic reticulum compartment.-   Park et al, J Biol Chem. 2004 Nov. 26; 279(48):50630-8. Epub 2004    Sep. 22, Post-transcriptional regulation of low density lipoprotein    receptor protein by proprotein convertase subtilisin/kexin type 9a    in mouse liver.-   Rashid et al, Proc Natl Acad Sci USA. 2005 Apr. 12; 102(15):5374-9.    Epub 2005 Apr. 1, Decreased plasma cholesterol and hypersensitivity    to statins in mice lacking Pcsk9.-   Kotowski et al, Am J Hum Genet. 2006 March; 78(3):410-22. Epub 2006    Jan. 20, A spectrum of PCSK9 alleles contributes to plasma levels of    low-density lipoprotein cholesterol.-   Chen et al, J Am Coll Cardiol. 2005 May 17; 45(10):1611-9. Epub 2005    Apr. 21, A common PCSK9 haplotype, encompassing the E670G coding    single nucleotide polymorphism, is a novel genetic marker for plasma    low-density lipoprotein cholesterol levels and severity of coronary    atherosclerosis.-   Pisciotta et al, Atherosclerosis. 2006 June; 186(2):433-40. Epub    2005 Sep. 23, Additive effect of mutations in LDLR and PCSK9 genes    on the phenotype of familial hypercholesterolemia.-   Zhao et al, Am J Hum Genet. 2006 September; 79(3):514-23. Epub 2006    Jul. 18, Molecular characterization of loss-of-function mutations in    PCSK9 and identification of a compound heterozygote.-   Seidah et al, Proc Natl Acad Sci USA. 2003 Feb. 4; 100(3):928-33.    Epub 2003 Jan. 27, The secretory proprotein convertase neural    apoptosis-regulated convertase 1 (NARC-1): liver regeneration and    neuronal differentiation.

Example 2 IL4 Receptor Alpha (IL4Ra; CD124)

Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1)was originally characterized by its ability to stimulate theproliferation of B cells in response to low concentrations of antibodiesdirected to surface immunoglobulin. IL-4 has been shown to possess abroad spectrum of biological activities, including growth stimulation ofT cells, mast cells, granulocytes, megakaryocytes and erythrocytes. IL-4induces the expression of class Il major histocompatibility complexmolecules in resting B cells, and enhances the secretion of IgE and IgGIisotypes by stimulated B cells

In an example, the invention provides a method of treating or reducingthe risk of an IL4Ra-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human IL4RAprotein. The invention also provides a corresponding ligand.

The present invention provides anti-IL4Ra ligands; and IL4Ra-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of IL4Ra, in particular human IL4Ra or its ligands and inscreening assays to identify other antagonists of IL4Ra activity. Someof the ligands of the invention are useful for inhibiting binding ofIL4Ra to IL4, or inhibiting IL4Ra-mediated activities.

Anti-IL4Ra ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human IL4Ra, which is a commonly-occurring form (see, eg, SEQ ID NO:67). While such therapies are useful for human patients harbouring thisform of human IL4RA, the inventor considered it useful to investigatethe possibility of targeting rarer—but still naturally-occurring—formsof IL4Ra amongst human populations. In this way, the inventor arrived atinsight into the natural occurrences and distributions of rarer humanIL4Ra forms that can serve as useful targets (at the protein or nucleicacid level) for human treatment, prophylaxis and diagnosis pertinent todiseases and conditions mediated or associated with IL4RA activity. Thisparticularly provides for tailored therapies, prophylaxis and diagnosisin humans that are devoid of the common IL4Ra gene or protein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer IL4Ra polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous changesat the nucleotide level that translate into one or more correspondingchanges in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor realised that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-IL4Ra ligand for administration to human patients fortherapy and/or prophylaxis of IL4Ra-mediated or associated diseases orconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the presentinventor decided to determine a set of human IL4Ra variants on the basisof the following criteria, these being criteria that the inventorrealised would provide for useful medical drugs and diagnostics totailored need in the human population. The inventor selected variantshaving at least 3 of the 4 following criteria:—

Naturally-occurring human IL4Ra variation having a cumulative humanallele frequency of 35% or less;

Naturally-occurring human IL4Ra variation having a total human genotypefrequency of about 50% or less;

Naturally-occurring human IL4Ra variation found in many different humanethnic populations (using the standard categorisation of the 1000Genomes Project; see Table 2 below); and

Naturally-occurring human IL4Ra variation found in many individualsdistributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listedin Table 11 below. The inventor's selection included, as an additionalor alternative consideration, selection for nucleotide variation thatproduced amino acid variation in corresponding IL4Ra forms (ie,non-synonymous variations), as opposed to silent variations that do notalter amino acid residues in the target protein.

In an example, the invention provides a method of treating or reducingthe risk of an IL4Ra-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human IL4RAprotein that comprises a mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Asexplained further below, these amino acid variations are found innaturally-occurring IL-4Ra variants in humans found in many populations.Said human comprises a nucleotide sequence encoding said IL4RA proteincomprising said mutation selected from the group consisting of I75V,E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

An example also provides a ligand (eg, an antibody or antibody fragment)for treating or reducing the risk of an IL4Ra-mediated disease orcondition in a human in need thereof, the method comprisingadministering to said human said ligand, wherein the ligand specificallybinds a human IL4RA protein that comprises a mutation selected from thegroup consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ IDNO: 67. Said human comprises a nucleotide sequence encoding said IL4RAprotein comprising said mutation selected from the group consisting of175V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In an example, the invention provides a method of targeting IL4Ra in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human IL4RAprotein that comprises a mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Said humancomprises a nucleotide sequence encoding said IL4RA protein comprisingsaid mutation selected from the group consisting of I75V, E400A, C431R,S503P, Q576R and S752A in SEQ ID NO: 67. In an example, the human issuffering from or at risk of an IL4Ra-mediated disease or condition. Inan example, the method treats or reduces the risk of an IL4Ra-mediateddisease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment)for targeting IL4Ra in a human, the method comprising administering tosaid human said ligand, wherein the ligand specifically binds a humanIL4RA protein that comprises a mutation selected from the groupconsisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO:67. Said human comprises a nucleotide sequence encoding said IL4RAprotein comprising said mutation selected from the group consisting ofI75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In anexample, the human is suffering from or at risk of an IL4Ra-mediateddisease or condition. In an example, the method treats or reduces therisk of an IL4Ra-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40; andwherein (ii) said human comprises a nucleotide sequence encoding saidIL4RA protein comprising said mutation selected from the groupconsisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO:67.

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises a nucleotide sequence encoding said IL4RA protein comprisingsaid mutation selected from the group consisting of I75V, E400A, C431R,S503P, Q576R and S752A in SEQ ID NO: 67.

The biological activities of IL-4 are mediated by specific cell surfacereceptors for IL-4. Human IL-4 receptor alpha (h1L-4Ra) is described in,for example, U.S. Pat. Nos. 5,599,905, 5,767,065, and 5,840,869.Antibodies to h1L-4R are described in U.S. Pat. No. 5,717,072.

Methods for using antibodies to h1L-4R are described in U.S. Pat. Nos.5,714,146; 5,985,280; and 6,716,587.

The human IL-4Ra (aka IL-4Ra) subunit (Swiss Prot accession numberP24394) is a 140 kDa type 1 membrane protein that binds human IL-4 witha high affinity (Andrews et al J. Biol. Chem (2002) 277:46073-46078).The IL-4/IL-4Ra complex can dimerize with either the common gamma chain(γc, CD132) or the IL-13Ralpha1 (IL-13Rα1) subunit, via domains on IL-4,to create two different signalling complexes, commonly referred to asType I and Type II receptors, respectively. Alternatively, IL-13 canbind IL-13Rα1 to form an IL-13/IL-13Rα1 complex that recruits the IL-4Rasubunit to form a Type II receptor complex. Thus, IL-4Ra mediates thebiological activities of both IL-4 and IL-13 (reviewed by Gessner et al,Immunobiology, 201:285, 2000). In vitro studies have shown that IL-4 andIL-13 activate effector functions in a number of cell types, for examplein T cells, B cells, eosinophils, mast cells, basophils, airway smoothmuscle cells, respiratory epithelial cells, lung fibroblasts, andendothelial cells (reviewed by Steinke et al, Resp Res, 2:66, 2001, andby Willis-Karp, Immunol Rev, 202:175, 2004).

IL-4 by binding to its receptor (IL-4R) is essential for the developmentof airway inflammation present in asthma, through the induction of IgEsynthesis in B cells and differentiation of T cells to a Th2 phenotype.Dupilumab is a monoclonal antibody designed for the treatment of atopicdiseases. It binds to the alpha subunit of the interleukin-4 receptor.Through blockade of IL-4R alpha, dupilumab modulates signaling of boththe IL-4 and IL-13 pathway, which have been implicated in thepathophysiology of allergic disease. Anti-IL4Ra ligands, antibodies andfragments according to the invention can, for example, be used fortreating or reducing the risk of allergic disease.

In an example, the ligand, antibody or fragment is for treating orreducing the risk of (or treats or reduces the risk of) allergic asthma,eosinophilic asthma or atopic dermatitis, optionally wherein theantibody is dupilumab.

In addition to its role in asthma, IL-4Ra has been linked with a numberof other pathologies, e.g. as follows (anti-IL4Ra ligands, antibodiesand fragments according to the invention can, for example, be used fortreating or reducing the risk of any one of these diseases orconditions):

Chronic Obstructive Pulmonary Disease (COPD) includes patientpopulations with varying degrees of chronic bronchitis, small airwaydisease and emphysema and is characterised by progressive irreversiblelung function decline that responds poorly to current asthma basedtherapy. The underlying causes of COPD remain poorly understood. The“Dutch hypothesis” proposes that there is a common susceptibility toCOPD and asthma and therefore, that similar mechanisms may contribute tothe pathogenesis of both disorders (Sluiter et al., Eur Respir J,).Zheng et al (J Clin Invest, 106(9):1081-93, 2000) have demonstrated thatoverexpression of IL-13 in the mouse lung caused emphysema, elevatedmucus production and inflammation, reflecting aspects of human COPD.Furthermore, AHR, an IL-13 dependent response in murine models ofallergic inflammation, has been shown to be predictive of lung functiondecline in smokers (Tashkin et al., Am J Respir Crit Care Med, 153(6 Pt1):1802-11, 1996). A link has also been established between an IL-13promoter polymorphism and susceptibility to develop COPD (Van Der PouwKraan et al., Genes Immun, 3(7): 436-9, 2002). The signs are thereforethat IL-4/IL-13 pathway, and in particular IL-13, plays an importantrole in the pathogenesis of COPD.

In addition to asthma, the IL-4111-13 pathway has been linked to otherfibrotic conditions, like systemic sclerosis (Hasegawa et al., JRheumatol, 24(2):328-32, 1997), pulmonary fibrosis (Hancock et al., Am JRespir Cell Mol Biol, 18(1): 60-5, 1998), parasite-induced liverfibrosis (Fallon et al., J Immunol, 164(5): 2585-91, 2000; Chiaramonteet al., J Clin Invest, 104(6): 777-85, 1999; Chiaramonte Hepatology34(2):273-82, 2001), and cystic fibrosis (Hauber et al. J. Cyst Fibr,2:189, 2003).

IL-4 and to some extent IL-13, are crucial for B cell mediatedactivities, such as B cell proliferation, immunoglobulin secretion, andexpression of FcepsilonR. Clinical applications of an IL-4Ra inhibitorinclude for example, use in allergy therapy to suppress IgE synthesis(including for example atopic dermatitis and food allergy), use intransplation therapy to prevent transplant rejection, as well assuppression of delayed-type hypersensitivity or contact hypersensitivityreactions.

Il-4R antagonists may also find use as adjuvants to allergyimmunotherapy and as vaccine adjuvants.

IL-4Ra polymorphisms in the human population have been described(reviewed by Gessner et al, Immunobiology, 201:285, 2000) andassociation with IgE levels or clinical atopy has been reported in somepopulations. For instance, V75R576 IL-4Ra is associated with allergicasthma and enhanced IL-4Ra function (Risma et al. J.Immunol.169(3):1604-1610, 2002).

Anti-IL4Ra ligands, antibodies and fragments according to the inventionoptionally neutralise IL-4Ra with high potency, for example as describedin more detail in the Examples of EP2604628. Neutralisation meansinhibition of a biological activity mediated by IL-4Ra. Ligands,antibodies and fragments according to the invention may neutralise oneor more activities mediated by IL-4Ra. The inhibited biological activityis likely mediated by prevention of IL-4Ra forming a signalling complexwith gamma chain (or IL-13Ra) and either of the associated solubleligands, e.g. IL-4 or IL-13.

Neutralisation of IL-4 or IL-13 signalling through its IL-4Ra containingreceptor complex may be measured by inhibition of IL-4 or IL-13stimulated TF-1 cell proliferation.

The ligand, antibody or fragment according to the invention is, forexample, dupilumab or any one disclosed in WO 08/054606 (Regeneron),EP2604628 (Medimmune), WO 01/92340 (immnunex) or WO 05/047331 (Immunex),the disclosures of which (including the sequences, eg, VH, VL and heavyand light chain sequences) are incorporated herein by reference forpotential inclusion in one or more claims herein. According to a furtheraspect of the ligand, antibody or fragment according to the invention iscapable of binding human interleukin-4 receptor alpha (hIL-4Ra) andcynomolgus monkey interleukin-4 receptor alpha (cyIL-4Ra). CynomolgusIL-4Ra cDNA sequence is shown as SEQ ID NO: 455 EP2604628. In aparticular embodiment the antibody or fragment is a human antibody orfragment. The ligand, antibody or fragment according to the invention isan antibody or fragment, for example, that comprises a VH and/or VL ofdupilumab, or comprises a heavy and/or light chain of dupilumab. In oneembodiment, the ligand, antibody or fragment comprises a VH domaincomprising SEQ ID NO: 69. In one embodiment, the ligand, antibody orfragment comprises a VL domain comprising SEQ ID NO: 70. In oneembodiment, the ligand, antibody or fragment comprises a heavy chaincomprising SEQ ID NO: 71. In one embodiment, the ligand, antibody orfragment comprises a light chain comprising SEQ ID NO: 72.

In a specific embodiment, the ligand, antibody or fragment of presentinvention comprises an Fc region, wherein the Fc region comprises atleast one non-native amino acid residue selected from the groupconsisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V,234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y,2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y,240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R. 243W, 243L 243Y,243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E,256M, 262I, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 264I, 264W,264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V,2651, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H,269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N,296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981, 298T,298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D,325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H,328A, 329F, 329H, 329Q, 330K, 330G, 3301, 330C, 330L, 330Y, 330V, 3301,330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E,331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331I, 332D, 332S,332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N,378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered by the EUindex as set forth in Kabat. Optionally, the Fc region may compriseadditional and/or alternative non-native amino acid residues known toone skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919; WO04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention is fortreating or preventing or reducing the risk of (or treats or prevents orreduces the risk of), for example, any disease or condition disclosed inany of WO 08/054606 (Regeneron), EP2604628 (Medimmune), WO 01/92340(immnunex) and WO 05/047331 (Immunex), the disclosures of which diseasesand conditions are incorporated herein by reference for potentialinclusion in one or more claims herein.

Further encompassed by the invention is the use of the ligand, antibodyor fragment in the manufacture of a medicament for use to attenuate orinhibit an IL-4Ra-mediated disease or disorder in a human.IL-4Ra-mediated or related disorders which are treated by the ligand,antibody or fragment of the invention include, for example, arthritis(including septic arthritis), herpetiformis, chronic idiopathicurticaria, scleroderma, hypertrophic scarring, Whipple's Disease, benignprostate hyperplasia, lung disorders, such as mild, moderate or severeasthma, inflammatory disorders such as inflammatory bowel disease,allergic reactions, Kawasaki disease, sickle cell disease, Churg-Strausssyndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome, autoimmunelymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett'sesophagus, autoimmune uveitis, tuberculosis, and nephrosis.

Particular conditions for which a ligand, antibody or fragment of theinvention may be used in treatment or diagnosis include: asthma, COPD(eg, chronic bronchitis, small airway disease or emphysema),inflammatory bowel disease, a fibrotic condition (eg, systemicsclerosis, pulmonary fibrosis, parasite-induced liver fibrosis, orcystic fibrosis), allergy (for example atopic dermatitis, dust miteallergy, pet allergy or food allergy), transplation therapy to preventtransplant rejection, suppression of a delayed-type hypersensitivity ora contact hypersensitivity reaction, as an adjuvant to allergyimmunotherapy or as a vaccine adjuvant. In an example, the method andligand are for treating or preventing (or reducing the risk of) nasalypolyps and/or sinusitis.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving IL-4, IL-13or IL-4Ra expression and/or activity. One embodiment, among others, is amethod of treatment comprising administering an effective amount of aligand, antibody or fragment of the invention to a patient in needthereof, wherein functional consequences of IL-4Ra activation aredecreased. Another embodiment, among others, is a method of treatmentcomprising (i) identifying a patient demonstrating IL-4, IL-13 or IL-4Raexpression or activity, and (ii) administering an effective amount of aligand, antibody or fragment of the invention to the patient, wherein afunctional consequence of IL-4Ra activation are attenuated. An effectiveamount according to the invention is an amount that modulates (e.g.decreases) the functional consequences of IL-4Ra activation so as tomodulate (e.g. decrease or lessen) the severity of at least one symptomof the particular disease or disorder being treated, but not necessarilycure the disease or disorder. Accordingly, one embodiment of theinvention is a method of treating or reducing the severity of at leastone symptom of any of the disorders referred to herein, comprisingadministering to a patient in need thereof an effective amount of one ormore ligands, antibodies or fragments of the present invention alone orin a combined therapeutic regimen with another appropriate medicamentknown in the art or described herein such that the severity of at leastone symptom of any of the disorders is reduced. Another embodiment ofthe invention, among others, is a method of antagonizing at least oneeffect of IL-4Ra comprising contacting with or administering aneffective amount of one or more ligands, antibodies or fragments of thepresent invention such that said at least one effect of IL-4Ra isantagonized, e.g. the ability of IL-4Ra to form a complex (the precursorto active signalling) with IL-4.

A ligand, antibody or fragment of the invention can, in an example, beused in combination with another therapeutic agent for the treatment ofcancer. Suitable agents to be used in combination include thosedisclosed in EP2604628 (eg, in paragaraph [0287]), which disclosure isincorporated herein by reference.

Tailoring Antibodies to Rarer IL4Ra Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1),the invention includes the possibility to tailor treatment of humansfurther by selecting antibody-based ligands with variable domains and/orconstant domains based on gene segments found in many humans of theethnic populations where the variant TOI forms are found to meet theselection criteria of the invention. This also applies mutatis mutandiswhere the TOI is human IL4Ra, as in the present example. Thus, alldisclosure herein relating to tailoring variable and/or constant domainsapply to the present example, relating to IL4Ra and is combinable foruse in one or more claims herein.

As described in Example 1, an example is provided for ligands comprisingantibody VH domains derived from recombination of human IGHV genesegments comprising selected nucleotides at positions in the HCDR1 orFW3 where there is variability in humans (ie, where SNPs occur inhumans).

Further information is provided in Table 4, which shows variation atthese positions, as well as the variant distributions across the 1000Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ or constant region gene segments (see further Table 9 forrepresentative variants).

In an example, following this guidance, the chosen ligand is dupilumab.

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4.(vi) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Valat position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shownin SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asnat position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ IDNO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257shown in SEQ ID NO: 6.(vii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(viii) wherein the ligand comprises a human IGLC1*01 lambda chainconstant region and wherein said human comprises (i) a human IGLC1*01lambda chain constant region gene segment, or the human expressesantibodies comprising human IGLC1*01 lambda chain constant regions.(ix) wherein the ligand comprises a human gamma-4 heavy chain constantregion that comprises a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73 and wherein said humancomprises (i) an IGHG4*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-4heavy chain constant regions comprising a Leu at position 189 shown inSEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73.

For example, as per example (ix), the inventor identified thepossibility of addressing IGH-gamma-4 variation and identified utilityfor variations 189L and 289R individually or in combination, since theseresidues are part of the CH3 domain, and as such they form part ofantibody Fc regions. Thus, matching of these CH3 variations with thepatient is especially beneficial for reasons as discussed above. Thus,in this example the ligand of the invention comprises or consists of anantibody that comprises a human gamma-4 heavy chain constant region thatcomprises a Leu corresponding to position 189 of SEQ ID NO: 73 or an Argcorresponding to position 289 of SEQ ID NO: 73 and wherein the genome ofthe human comprises a gamma-4 heavy chain constant region nucleotidesequence that encodes such a Leu and/or Arg or the human expressesantibodies comprising human gamma-4 constant regions comprising such aLeu and/or Arg. An example of such a ligand is dupilumab.

Determination of Specific Binding of Ligands of the Invention to IL4RAVariants

The specific binding of Ligands of the invention to IL4Ra variants canbe performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of an IL4Ra-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human IL4Ra protein    that comprises a mutation selected from the group consisting of    I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an alternative, clause 1 provides:—    -   A method of targeting IL4Ra in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human IL4Ra protein        that comprises a mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an example, the human is suffering from or at risk of an        IL4Ra-mediated disease or condition.    -   In an example, the method treats or reduces the risk of an        IL4Ra-mediated disease or condition in the human. Optionally the        disease is asthma. Optionally the disease is atopic dermatitis.        Optionally the disease is nasal polyps and/or sinusitis.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation 175V.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation E400A.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation C431R.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation S503P.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation Q576R.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation S752A.-   2. The method of clause 1, wherein the IL4Ra comprises mutations    175V and Q576R in SEQ ID NO: 67 and optionally the disease is    asthma. Optionally the disease is atopic dermatitis. Optionally the    disease is nasal polyps and/or sinusitis.    -   Q576R and I75V are associated with airway inflammation disease,        eg, asthma. For example, see Clin Exp Allergy. 2003 August;        33(8):1111-7, “Polymorphisms in the interleukin-4 and        interleukin-4 receptor alpha chain genes confer susceptibility        to asthma and atopy in a Caucasian population”, Beghé B et al        (referring to I75V instead as I50V); J Asthma. 2010 April;        47(3):238-44. doi: 10.3109/02770900903509099, “Association and        gene-gene interactions of eight common single-nucleotide        polymorphisms with pediatric asthma in middle china”, Wu X et        al; Clin Exp Allergy. 2004 October; 34(10):1570-5, “Haplotypes        of the interleukin-4 receptor alpha chain gene associate with        susceptibility to and severity of atopic asthma”, Hytönen A M et        al, all incorporated herein by reference.-   3. The method of clause 1 or 2, wherein the nucleotide sequence    of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).    -   This variation is associated with airway inflammation disease,        eg, asthma.-   4. The method of any preceding clause, wherein each said human VH    gene segment comprises SEQ ID NO: 39.-   5. The method of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes an IL4Ra    protein comprising said mutation selected from the group consisting    of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67    and/or an IL4Ra protein comprising said mutation selected from the    group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in    SEQ ID NO: 67.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes an IL4Ra protein comprising said mutation selected from    the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A    in SEQ ID NO: 67 and/or (b) an IL4Ra protein comprising said    mutation said mutation selected from the group consisting of I75V,    E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a IL4Ra protein comprising said mutation selected    from the group consisting of I75V, E400A, C431R, S503P, Q576R and    S752A in SEQ ID NO: 67.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding an        IL4Ra protein comprising said mutation selected from the group        consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ        ID NO: 67 or comprising an antisense sequence of said contiguous        nucleotides, wherein said sequence of contiguous nucleotides        comprises a nucleotide sequence encoding said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67, thereby forming a complex when at        least one nucleotide sequence encoding the IL4Ra protein        comprising said mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the IL4Ra protein comprising said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 68, or said human    is indicated as homozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to an asthma    treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received an asthma treatment or has reduced    responsiveness to an asthma treatment.-   16. The method of any preceding clause, wherein said disease or    condition is an inflammatory disease or condition; an atopic disease    or condition; a respiratory disease or condition; a disease or    condition associated with elevated IgE; or a disease or condition    associated with elevated IL-4 and/or IL-13 activity.-   17. The method of any preceding clause, wherein said disease or    condition is selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition selected from the group    consisting of an airway inflammatory disease or condition, chronic    obstructive pulmonary disease, asthma, pneumonia, hypersensitivity    pneumonitis, pulmonary infiltrate with eosinophilia, environmental    lung disease, pneumonia, bronchiectasis, cystic fibrosis,    interstitial lung disease, primary pulmonary hypertension, pulmonary    thromboembolism, disorders of the pleura, disorders of the    mediastinum, disorders of the diaphragm, hypoventilation,    hyperventilation, sleep apnea, acute respiratory distress syndrome,    mesothelioma, sarcoma, graft rejection, graft versus host disease,    lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,    aspergillosis, bronchiectasis, chronic bronchitis, emphysema,    eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive    pneumococcal disease, influenza, nontuberculous mycobacteria,    pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,    pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary    anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,    pulmonary histiocytosis X, pulmonary hypertension, pulmonary    nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive    disease, rheumatoid lung disease, sarcoidosis, and Wegener's    granulomatosis.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of at    least one condition selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275    and rs1805016.-   21. The method of any preceding clause, wherein said antibody or    antibody fragment is administered by inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.-   22. The method of any preceding clause, wherein the VH domain    comprises the amino acid sequence of SEQ ID NO: 69.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of an IL4Ra-mediated disease    or condition (eg, asthma) in a human in need thereof, wherein the    ligand specifically binds a human IL4RA protein that comprises a    mutation selected from the group consisting of I75V, E400A, C431R,    S503P, Q576R and S752A in SEQ ID NO: 67; wherein (i) the ligand    comprises a VH domain derived from the recombination of a human VH    segment, a human D gene segment and a human JH segment, the human VH    segment encoding the framework 1 of SEQ ID NO: 40 and wherein said    human comprises a VH gene segment encoding the framework 1 of SEQ ID    NO: 40, or the human expresses VH domains that comprise the    framework 1 of SEQ ID NO: 40; and wherein (ii) said human comprises    a nucleotide sequence encoding said IL4RA protein comprising said    mutation selected from the group consisting of I75V, E400A, C431R,    S503P, Q576R and S752A in SEQ ID NO: 67.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting IL4Ra in a human, wherein the ligand        specifically binds a human IL4RA protein that comprises a        mutation selected from the group consisting of I75V, E400A,        C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an example, the human is suffering from or at risk of an        IL4Ra-mediated disease or condition.    -   In an example, the method treats or reduces the risk of an        IL4Ra-mediated disease or condition in the human.-   2. The ligand of paragraph 1, wherein the IL4Ra comprises mutations    175V and Q576R in SEQ ID NO: 67 and optionally the disease is    asthma.    -   The ligand of paragraph 1 or 2, wherein the nucleotide sequence        of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).

The ligand of any preceding paragraph, wherein each said human VH genesegment comprises SEQ ID NO: 39.

-   3. The ligand of any preceding paragraph, wherein the VH gene    segment comprised by said human is a germline VH gene segment.-   4. The ligand of any preceding paragraph, said method comprising,    before said administering, selecting a human comprising said    nucleotide sequence of (ii), wherein the human is the human of    paragraph 1.-   5. The ligand of any preceding paragraph, wherein the human has been    determined to comprise the nucleotide sequence that encodes an IL4Ra    protein comprising said mutation selected from the group consisting    of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67    and/or an IL4Ra protein comprising said mutation selected from the    group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in    SEQ ID NO: 67.-   6. The ligand of any preceding paragraph, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes an IL4Ra protein comprising said mutation    selected from the group consisting of I75V, E400A, C431R, S503P,    Q576R and S752A in SEQ ID NO: 67 and/or (b) an IL4Ra protein    comprising said mutation said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein the determining step is performed before    administration of the antibody to the human.-   7. The ligand of paragraph 8, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding a IL4Ra protein comprising said    mutation selected from the group consisting of I75V, E400A, C431R,    S503P, Q576R and S752A in SEQ ID NO: 67.-   8. The ligand of paragraph 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding an        IL4Ra protein comprising said mutation selected from the group        consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ        ID NO: 67 or comprising an antisense sequence of said contiguous        nucleotides, wherein said sequence of contiguous nucleotides        comprises a nucleotide sequence encoding said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67, thereby forming a complex when at        least one nucleotide sequence encoding the IL4Ra protein        comprising said mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the IL4Ra protein comprising said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67.-   9. The ligand of paragraph 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   10. The ligand of paragraph 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   11. The ligand of any preceding paragraph, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 68, or said human    is indicated as homozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67.-   12. The ligand of any preceding paragraph, wherein said human is or    has been further determined to be substantially resistant to an    asthma treatment.-   13. The ligand of any preceding paragraph, wherein said human is    receiving or has received an asthma treatment or has reduced    responsiveness to an asthma treatment.-   14. The ligand of any preceding paragraph, wherein said disease or    condition is an inflammatory disease or condition; an atopic disease    or condition; a respiratory disease or condition; a disease or    condition associated with elevated IgE; or a disease or condition    associated with elevated IL-4 and/or IL-13 activity.-   15. The ligand of any preceding paragraph, wherein said disease or    condition is selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis.-   16. The ligand of any preceding paragraph, wherein said human has    been diagnosed with at least one condition selected from the group    consisting of an airway inflammatory disease or condition, chronic    obstructive pulmonary disease, asthma, pneumonia, hypersensitivity    pneumonitis, pulmonary infiltrate with eosinophilia, environmental    lung disease, pneumonia, bronchiectasis, cystic fibrosis,    interstitial lung disease, primary pulmonary hypertension, pulmonary    thromboembolism, disorders of the pleura, disorders of the    mediastinum, disorders of the diaphragm, hypoventilation,    hyperventilation, sleep apnea, acute respiratory distress syndrome,    mesothelioma, sarcoma, graft rejection, graft versus host disease,    lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,    aspergillosis, bronchiectasis, chronic bronchitis, emphysema,    eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive    pneumococcal disease, influenza, nontuberculous mycobacteria,    pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,    pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary    anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,    pulmonary histiocytosis X, pulmonary hypertension, pulmonary    nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive    disease, rheumatoid lung disease, sarcoidosis, and Wegener's    granulomatosis.-   17. The ligand of any preceding paragraph, wherein said antibody or    antibody fragment treats or reduces the risk in said human of at    least one condition selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis-   18. The ligand of any preceding paragraph, wherein the nucleotide    sequence comprises one or more

SNPs selected from the group consisting of rs1805010, rs1805011,rs1805012, rs1805015, rs1801275 and rs1805016.

-   19. The ligand of any preceding paragraph, wherein said antibody or    antibody fragment is for inhaled, intravenous or subcutaneous    administration and/or is comprised in an inhalable or injectable    preparation.-   20. The ligand of any preceding paragraph, wherein the VH domain    comprises the amino acid sequence of SEQ ID NO: 69.

Methods with C Region Tailoring

-   1. A method of treating or reducing the risk of an IL4Ra-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human IL4Ra protein    that comprises a mutation selected from the group consisting of    I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an alternative, clause 1 provides:—    -   A method of targeting IL4Ra in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human IL4Ra protein        that comprises a mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii)        said human comprises a nucleotide sequence encoding said IL4RA        protein comprising said mutation selected from the group        consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ        ID NO: 67.    -   In an example, the human is suffering from or at risk of an        IL4Ra-mediated disease or condition.    -   In an example, the method treats or reduces the risk of an        IL4Ra-mediated disease or condition in the human.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation 175V.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation E400A.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation C431R.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation S503P.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation Q576R.    -   In an example of the method or ligand of the invention, the        IL4Ra comprises mutation S752A. 2. The method of clause 1,        wherein the IL4Ra comprises mutations 175V and Q576R in SEQ ID        NO: 67 and optionally the disease is asthma.-   3. The method of clause 1 or 2, wherein the nucleotide sequence    of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).-   4. The method of any preceding clause, wherein said human gamma-4    heavy chain constant region that comprises SEQ ID NO: 73.-   5. The method of any preceding clause, wherein the human expresses    antibodies comprising human IGHG4*01 heavy chain constant regions.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes an IL4Ra    protein comprising said mutation selected from the group consisting    of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67    and/or an IL4Ra protein comprising said mutation selected from the    group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in    SEQ ID NO: 67.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes an IL4Ra protein comprising said mutation selected from    the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A    in SEQ ID NO: 67 and/or (b) an IL4Ra protein comprising said    mutation said mutation selected from the group consisting of I75V,    E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a IL4Ra protein comprising said mutation selected    from the group consisting of I75V, E400A, C431R, S503P, Q576R and    S752A in SEQ ID NO: 67.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding an        IL4Ra protein comprising said mutation selected from the group        consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ        ID NO: 67 or comprising an antisense sequence of said contiguous        nucleotides, wherein said sequence of contiguous nucleotides        comprises a nucleotide sequence encoding said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67, thereby forming a complex when at        least one nucleotide sequence encoding the IL4Ra protein        comprising said mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is        present; and detecting the presence or absence of the complex,        wherein detecting the presence of the complex determines that        the human comprises the IL4Ra protein comprising said mutation        selected from the group consisting of I75V, E400A, C431R, S503P,        Q576R and S752A in SEQ ID NO: 67.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 68, or said human    is indicated as homozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to an asthma    treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received an asthma treatment or has reduced    responsiveness to an asthma treatment.-   16. The method of any preceding clause, wherein said disease or    condition is an inflammatory disease or condition; an atopic disease    or condition; a respiratory disease or condition; a disease or    condition associated with elevated IgE; or a disease or condition    associated with elevated IL-4 and/or IL-13 activity.-   17. The method of any preceding clause, wherein said disease or    condition is selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition selected from the group    consisting of an airway inflammatory disease or condition, chronic    obstructive pulmonary disease, asthma, pneumonia, hypersensitivity    pneumonitis, pulmonary infiltrate with eosinophilia, environmental    lung disease, pneumonia, bronchiectasis, cystic fibrosis,    interstitial lung disease, primary pulmonary hypertension, pulmonary    thromboembolism, disorders of the pleura, disorders of the    mediastinum, disorders of the diaphragm, hypoventilation,    hyperventilation, sleep apnea, acute respiratory distress syndrome,    mesothelioma, sarcoma, graft rejection, graft versus host disease,    lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,    aspergillosis, bronchiectasis, chronic bronchitis, emphysema,    eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive    pneumococcal disease, influenza, nontuberculous mycobacteria,    pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,    pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary    anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,    pulmonary histiocytosis X, pulmonary hypertension, pulmonary    nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive    disease, rheumatoid lung disease, sarcoidosis, and Wegener's    granulomatosis.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of at    least one condition selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275    and rs1805016.-   21. The method of any preceding clause, wherein said antibody or    antibody fragment is administered by inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.    Ligands with Constant Region Tailoring-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of an IL4Ra-mediated disease    or condition (eg, asthma) in a human in need thereof, wherein the    ligand specifically binds a human IL4RA protein that comprises a    mutation selected from the group consisting of I75V, E400A, C431R,    S503P, Q576R and S752A in SEQ ID NO: 67; wherein (i) the ligand    comprises a human gamma-4 heavy chain constant region that comprises    a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at position    289 shown in SEQ ID NO: 73 and wherein said human comprises (i) an    IGHG4*01 human heavy chain constant region gene segment, or the    human expresses antibodies comprising human gamma-4 heavy chain    constant regions comprising a Leu at position 189 shown in SEQ ID    NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting IL4Ra in a human, wherein the ligand        specifically binds a human IL4RA protein that comprises a        mutation selected from the group consisting of I75V, E400A,        C431R, S503P, Q576R and S752A in SEQ ID NO: 67;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said IL4RA protein comprising said mutation selected from the        group consisting of I75V, E400A, C431R, S503P, Q576R and S752A        in SEQ ID NO: 67.    -   In an example, the human is suffering from or at risk of an        IL4Ra-mediated disease or condition. In an example, the method        treats or reduces the risk of an IL4Ra-mediated disease or        condition in the human.-   2. The ligand of paragraph 1, wherein the IL4Ra comprises mutations    175V and Q576R in SEQ ID NO: 67 and optionally the disease is    asthma.-   3. The ligand of paragraph 1 or 2, wherein the nucleotide sequence    of (ii) comprises nucleotide mutation −3223T (dB SNP numbering).-   4. The ligand of any preceding paragraph, wherein said human gamma-4    heavy chain constant region that comprises SEQ ID NO: 73.-   5. The ligand of any preceding paragraph, wherein the human    expresses antibodies comprising human IGHG4*01 heavy chain constant    regions.-   6. The ligand of any preceding paragraph, said method comprising,    before said administering, selecting a human comprising said    nucleotide sequence of (ii), wherein the human is the human of    paragraph 1.-   7. The ligand of any preceding paragraph, wherein the human has been    determined to comprise the nucleotide sequence that encodes an IL4Ra    protein comprising said mutation selected from the group consisting    of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67    and/or an IL4Ra protein comprising said mutation selected from the    group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in    SEQ ID NO: 67.-   8. The ligand of any preceding paragraph, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes an IL4Ra protein comprising said mutation    selected from the group consisting of I75V, E400A, C431R, S503P,    Q576R and S752A in SEQ ID NO: 67 and/or (b) an IL4Ra protein    comprising said mutation said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The ligand of paragraph 8, wherein the step of determining    comprises assaying a biological sample from the human for a    nucleotide sequence encoding a IL4Ra protein comprising said    mutation selected from the group consisting of I75V, E400A, C431R,    S503P, Q576R and S752A in SEQ ID NO: 67.-   10. The ligand of paragraph 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding an        IL4Ra protein comprising said mutation selected from the group        consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ        ID NO: 67 or comprising an antisense sequence of said contiguous        nucleotides, wherein said sequence of contiguous nucleotides        comprises a nucleotide sequence encoding said mutation selected        from the group consisting of I75V, E400A, C431R, S503P, Q576R        and S752A in SEQ ID NO: 67, thereby forming a complex when at        least one nucleotide sequence encoding the IL4Ra protein        comprising said mutation selected from the group consisting of        I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex    -   determines that the human comprises the IL4Ra protein comprising        said mutation selected from the group consisting of I75V, E400A,        C431R, S503P, Q576R and S752A in SEQ ID NO: 67.-   11. The ligand of paragraph 9, wherein the assaying comprises    nucleic acid amplification and optionally one or more methods    selected from sequencing, next generation sequencing, nucleic acid    hybridization, and allele-specific amplification and/or wherein the    assaying is performed in a multiplex format.-   12. The ligand of paragraph 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding paragraph, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67, optionally, wherein said human is further indicated as    comprising the nucleotide sequence of SEQ ID NO: 68, or said human    is indicated as homozygous for a nucleotide sequence encoding the    IL4Ra protein comprising said mutation selected from the group    consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID    NO: 67.-   14. The ligand of any preceding paragraph, wherein said human is or    has been further determined to be substantially resistant to an    asthma treatment.-   15. The ligand of any preceding paragraph, wherein said human is    receiving or has received an asthma treatment or has reduced    responsiveness to an asthma treatment.-   16. The ligand of any preceding paragraph, wherein said disease or    condition is an inflammatory disease or condition; an atopic disease    or condition; a respiratory disease or condition; a disease or    condition associated with elevated IgE; or a disease or condition    associated with elevated IL-4 and/or IL-13 activity.-   17. The ligand of any preceding paragraph, wherein said disease or    condition is selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis.-   18. The ligand of any preceding paragraph, wherein said human has    been diagnosed with at least one condition selected from the group    consisting of an airway inflammatory disease or condition, chronic    obstructive pulmonary disease, asthma, pneumonia, hypersensitivity    pneumonitis, pulmonary infiltrate with eosinophilia, environmental    lung disease, pneumonia, bronchiectasis, cystic fibrosis,    interstitial lung disease, primary pulmonary hypertension, pulmonary    thromboembolism, disorders of the pleura, disorders of the    mediastinum, disorders of the diaphragm, hypoventilation,    hyperventilation, sleep apnea, acute respiratory distress syndrome,    mesothelioma, sarcoma, graft rejection, graft versus host disease,    lung cancer, allergic rhinitis, allergy, asbestosis, aspergilloma,    aspergillosis, bronchiectasis, chronic bronchitis, emphysema,    eosinophilic pneumonia, idiopathic pulmonary fibrosis, invasive    pneumococcal disease, influenza, nontuberculous mycobacteria,    pleural effusion, pneumoconiosis, pneumocytosis, pneumonia,    pulmonary actinomycosis, pulmonary alveolar proteinosis, pulmonary    anthrax, pulmonary edema, pulmonary embolus, pulmonary inflammation,    pulmonary histiocytosis X, pulmonary hypertension, pulmonary    nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive    disease, rheumatoid lung disease, sarcoidosis, and Wegener's    granulomatosis.-   19. The ligand of any preceding paragraph, wherein said antibody or    antibody fragment treats or reduces the risk in said human of at    least one condition selected from the group consisting of an airway    inflammatory disease or condition, chronic obstructive pulmonary    disease, asthma, pneumonia, hypersensitivity pneumonitis, pulmonary    infiltrate with eosinophilia, environmental lung disease, pneumonia,    bronchiectasis, cystic fibrosis, interstitial lung disease, primary    pulmonary hypertension, pulmonary thromboembolism, disorders of the    pleura, disorders of the mediastinum, disorders of the diaphragm,    hypoventilation, hyperventilation, sleep apnea, acute respiratory    distress syndrome, mesothelioma, sarcoma, graft rejection, graft    versus host disease, lung cancer, allergic rhinitis, allergy,    asbestosis, aspergilloma, aspergillosis, bronchiectasis, chronic    bronchitis, emphysema, eosinophilic pneumonia, idiopathic pulmonary    fibrosis, invasive pneumococcal disease, influenza, nontuberculous    mycobacteria, pleural effusion, pneumoconiosis, pneumocytosis,    pneumonia, pulmonary actinomycosis, pulmonary alveolar proteinosis,    pulmonary anthrax, pulmonary edema, pulmonary embolus, pulmonary    inflammation, pulmonary histiocytosis X, pulmonary hypertension,    pulmonary nocardiosis, pulmonary tuberculosis, pulmonary    veno-occlusive disease, rheumatoid lung disease, sarcoidosis, and    Wegener's granulomatosis-   20. The ligand of any preceding paragraph, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs1805010, rs1805011, rs1805012, rs1805015, rs1801275    and rs1805016.-   21. The ligand of any preceding paragraph, wherein said antibody or    antibody fragment is for inhaled, intravenous or subcutaneous    administration and/or is comprised in an inhalable or injectable    preparation.

In an example of any other example or embodiment, clause or paragraphrelating to IL4Ra, the IL4Ra mutation is 75V and the human is of AFR orASN ancestry, eg, the ancestry of the human is selected from ASW, LWK,CHS or JPT. All of these ancestries have greater than the average of 47%frequency for 75V.

In an example of any other example or embodiment, clause or paragraphrelating to IL4Ra, the IL4Ra mutation is 400A and the human is of AFRancestry, eg, the ancestry of the human is selected from ASW, LWK andYRI. All of these ancestries have greater than the average frequency for400A.

In an example of any other example or embodiment, clause or paragraphrelating to IL4Ra, the IL4Ra mutation is 431R and the human is of AFR orAMR ancestry, eg, the ancestry of the human is selected from YRI, CLM,MXL, PUR or CEU. All of these ancestries have greater than the averagefrequency for 431R.

In an example of any other example or embodiment, clause or paragraphrelating to IL4Ra, the IL4Ra mutation is 752A and the human is of AFRancestry, eg, the ancestry of the human is selected from ASW, LWK andYRI. All of these ancestries have 37-39% frequency, which is greaterthan the average frequency for 752A.

Frequencies are with reference to the 1000 Genomes database (version20110521) here.

In an example of any other example or embodiment, clause or paragraphrelating to IL4Ra, the IL4Ra mutation is 75V and the human is of AFR orASN ancestry, eg, the ancestry of the human is selected from ASW, LWK,CHS or JPT. All of these ancestries have greater than the average of 47%frequency for 75V.

Example 3 Nav1.7 (SCN9A; ETHA; FEB3B; GEFSP7; NE-NA; NENA; PN1; SFNP)

Reference is made to J Clin Invest. 2007 December; 117(12):3603-9,“Mutations in sodium-channel gene SCN9A cause a spectrum of humangenetic pain disorders”, Drenth JP & Waxman S G (incorporated herein byreference). Voltage-gated sodium channels play a critical role in thegeneration and conduction of action potentials and are thus importantfor electrical signalling by most excitable cells. Sodium channels areintegral membrane proteins and are comprised of a large a subunit, whichforms the voltage-sensitive and ion-selective pore, and smallerauxiliary 13 subunit(s) that can modulate the kinetics and voltagedependence of channel gating. To date, we know of 9 isoforms of thesodium-channel a subunit (Nav1.1-Nav1.9), each with a unique central andperipheral nervous system distribution. Four closely related sodiumchannels (Nav1.1, -1.2, -1.3, and -1.7) are encoded by a set of 4 genes(SCNJA, SCN2A, SCN3A, and SCN9A, respectively) located within a clusteron chromosome 2q24.3. Nav1.7 has a critical role in pain sensation.Furthermore, from KO studies and animal pain models, it would appearthat Nav1.7 plays a major role in inflammatory pain.

Nav1.7 is located in peripheral neurons and plays an important role inaction potential production in these cells. Recent genetic studies haveidentified Nav1.7 dysfunction in three different human pain disorders.Gain-of-function missense mutations in Nav1.7 have been shown to causeprimary erythermalgia (PE) and paroxysmal extreme pain disorder (PEPD),while nonsense mutations in Nav1.7 result in loss of Nav1.7 function anda condition known as channelopathy-associated insensitivity to pain(CIP), a rare disorder in which affected individuals are unable to feelphysical pain. Recent work has shown that different types ofchannelopathies (diseases caused by disturbed function of ion channelsubunits or the proteins that regulate them), all involving the sameNav1.7 sodium channel, underlie these disorders.

Nav1.7 is encoded by SCN9A, a 113.5-kb gene comprising 26 exons (OMIM603415) (FIG. 1A, Drenth & Waxman). The encoded sodium channel iscomposed of 1977 amino acids organized into 4 domains, each with 6transmembrane segments, and is predominantly expressed in the dorsalroot ganglion (DRG) neurons and sympathetic ganglion neurons (FIG. 1B,Drenth & Waxman). Immunohistochemical studies show that Nav1.7 ispresent at the distal ends of the wire-like projections of neurons knownas neurites, close to the impulse trigger zone where neuronal firing isinitiated. Interestingly, the large majority of DRG neurons that expressNav1.7 are pain sensing (nociceptive), suggesting a role for this sodiumchannel in the pathogenesis of pain.

Cell. 2014 Jun. 5; 157(6):1393-404. doi: 10.1016/j.ce11.2014.03.064.Epub 2014 May 22; “A monoclonal antibody that targets a NaV1.7 channelvoltage sensor for pain and itch relief”, Lee J H et al and 2011 (bothincorporated herein by reference) describe the generation of amonoclonal antibody against huma Nav1.7.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.7-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a huma Nav1.7protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.7 ligands; and Nav1.7-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of Nav1.7, in particular huma Nav1.7 or its ligands and inscreening assays to identify other antagonists of Nav1.7 activity. Someof the ligands of the invention are useful for inhibitingNav1.7-mediated activities.

Anti-Nav1.7 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type” humaNav1.7, which is a commonly-occurring form (see, eg, SEQ ID NO: 75).While such therapies are useful for human patients harbouring this formof huma Nav1.7, the inventor considered it useful to investigate thepossibility of targeting rarer—but still naturally-occurring—forms ofNav1.7 amongst human populations. In this way, the inventor arrived atinsight into the natural occurrences and distributions of rarer humaNav1.7 forms that can serve as useful targets (at the protein or nucleicacid level) for human treatment, prophylaxis and diagnosis pertinent todiseases and conditions mediated or associated with NAV1.7 activity.This particularly provides for tailored therapies, prophylaxis anddiagnosis in humans that are devoid of the common Nav1.7 gene orprotein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer Nav1.7 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous (aka“missense”) changes at the nucleotide level that translate into one ormore corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor saw that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-Nav1.7 ligand for administration to human patientsfor therapy and/or prophylaxis of Nav1.7-mediated or associated diseasesor conditions. In this way, the patient receives drugs and ligands thatare tailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the presentinventor decided to determine a set of huma Nav1.7 variants on the basisof the following criteria, these being criteria that the inventorrealised would provide for useful medical drugs and diagnostics totailored need in the human population. The inventor selected variantshaving at least 3 of the 4 following criteria:—

Naturally-occurring huma Nav1.7 variation having a cumulative humanallele frequency of 35% or less;

Naturally-occurring huma Nav1.7 variation having a total human genotypefrequency of about 50% or less;

Naturally-occurring huma Nav1.7 variation found in many different humanethnic populations (using the standard categorisation of the 1000Genomes Project; see Table 2 below); and

Naturally-occurring huma Nav1.7 variation found in many individualsdistributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listedin Table 12 (see Further Variants) below. The inventor's selectionincluded, as an additional or alternative consideration, selection fornucleotide variation that produced amino acid variation in correspondingNav1.7 forms (ie, non-synonymous variations), as opposed to silentvariations that do not alter amino acid residues in the target protein.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.7-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a huma Nav1.7protein that comprises an amino acid selected from the group consistingof 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an aminoacid other than R), 328X (wherein X is an amino acid other than Y),395K, 459X (wherein X is an amino acid other than S), 693 X (wherein Xis an amino acid other than E), 767X (wherein X is an amino acid otherthan I), 830X (wherein X is an amino acid other than R), 897X (wherein Xis an amino acid other than W), 1200L, 1235L, 1488X (wherein X is anamino acid other than R), 1659X (wherein X is an amino acid other thanK), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G. As explained further below, these amino acidvariations are found in naturally-occurring Nav1.7 variants in humansfound in many populations. Said human comprises a nucleotide sequenceencoding said Nav1.7 protein comprising said amino acid selected fromthe group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693 X (wherein X is an amino acid other than E), 767X (wherein X isan amino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G.

For example, said amino acid is selected from the group consisting ofany one of (a) to (e):—

-   -   a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; or    -   b) 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; or    -   c) 277X (wherein X is an amino acid other than R), 328X (wherein        X is an amino acid other than Y), 395K, 459X (wherein X is an        amino acid other than S), 693 X (wherein X is an amino acid        other than E), 767X (wherein X is an amino acid other than I),        830X (wherein X is an amino acid other than R), 897X (wherein X        is an amino acid other than W), 1200L, 1235L, 1488X (wherein X        is an amino acid other than R), 1659X (wherein X is an amino        acid other than K) and 1689X (wherein X is an amino acid other        than W); or    -   d) 395K, 1200L and 1235L; or    -   e) 422D, 490N, 943L, 1002L, 1161W and 1919G.        Amino acids (a)-(d) are discussed in Drenth & Waxman as being        associated with pain conditions (either undesirable elevated or        reduced pain). Thus, in one embodiment, the disease or condition        is a pain disease or condition and the amino acid is selected        from (a), (b), (c) or (d). Thus, in one embodiment, the disease        or condition is an itching disease or condition and the amino        acid is selected from (a), (b), (c) or (d). In an example, the        disease is PE and the amino acid is selected from (a). In an        example, the disease is PEPD and the amino acid is selected from        (b). In an example, the disease is CIP and the amino acid is        selected from (c) or (d). In an example, the disease or        condition is a pain or itching disease or condition and the        amino acid is selected from (e).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathicpain. For example, the pain is painful diabetic neuropathy (PDN),post-herpetic neuropathy (PHN) or trigeminal neuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosispain, phantom limb pain, post-stroke pain, chronic back pain,osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronicinflammatory pain.

In one embodiment, the disease or condition is a channelopathy orassociated with a channelopathy.

In one embodiment, the disease or condition is selected from the groupconsisting of primary erythermalgia (PE), paroxysmal extreme paindisorder (PEPD) and channelopathy-associated insensitivity to pain(CIP).

Optionally, the NAV-mediated disease or condition is selected from anyone of the following:

-   -   a. Neuropathic or neurogenic pain (for example arising from        painful diabetic neuropathy (PDN), post-herpetic neuropathy        (PHN), central neuropathy, peripheral neuropathy, trigeminal        neuralgia (TN), anaesthesia dolorosa, spinal cord injuries,        multiple sclerosis, phantom limb pain, hyperalgesia,        hyperpathia, paresthesia, psychogenic pain, post-stroke pain and        HIV-associated pain, back pain, chronic back pain,        osteoarthritis, cancer, breakthrough pain, erythromelalgia [e.g.        primary erythromelalgia], paroxysmal 30 extreme pain disorder,        nerve compression and/or entrapment such as carpal tunnel        syndrome, tarsal tunnel syndrome, ulnar nerve entrapment,        compression radiculopathy, radicular low back pain, spinal root        lesions, spinal root compression, lumbar spinal stenosis,        sciatic nerve compression, intercostal neuralgia], neuritis,        pain from chemotherapy, congenital defect/channelopathy [e g        channelopathy-associated insensitivity to pain and congenital        insensitivity to pain] or chronic alcoholism [alcoholic        polyneuropathy]);    -   b. inflammatory pain (such as osteoarthritis pain, chronic back        pain, rheumatoid arthritis pain, cancer pain, breakthrough pain,        pain associated with burns, encephalitis pain, bone fracture        pain, neuritis pain, autoimmune disease pain, postoperative        pain, dental pain, pain associated with bacterial infection,        pain associated with radiotherapy, pain associated with gout and        irritable bowel syndrome pain);    -   c. pain from trauma (such as from lacerations, incisions, burns,        foreign bodies or bullet and/or shrapnel injuries, spinal cord        injury, brachial plexus avulsion, nerve crush and/or entrapment        (such as carpal tunnel syndrome, tarsal tunnel syndrome, ulnar        nerve entrapment, compression radiculopathy, radicular low back        pain, spinal root lesions, spinal root compression, lumbar        spinal stenosis, sciatic nerve compression, intercostal        neuralgia), nerve transection, post-operative pain, dental pain        and toxic exposure);    -   d. pain from infection (such as post-herpetic neuropathy (PHN),        HIV-associated pain small pox infection, encephalitis, herpes        infection, and bacterial infection);    -   e. pain from malignancy (such as cancer pain, breakthrough pain,        and nerve compression pain);    -   f. visceral pain (such as renal or ureteral colic, irritable        bowel syndrome, angina or cardiac pain, cardiac arrhythmia,        period pain, interstitial cystitis, rectal pain, pain associated        with diarrhoea, appendicitis, cholecystitis and pancreatitis);    -   g. metabolic or chronic disease pain (such as associated with        multiple sclerosis, cancer pain, breakthrough pain, pain        associated with gout, peripheral diabetic neuropathy, pain        associated with chronic alcoholism [alcoholic polyneuropathy],        uremia pain, pain associated with hypothyroidism and pain        associated with vitamin deficiency);    -   h. headache pain (such as tension headache, migraine and cluster        headache);    -   i. idiopathic pain (such as trigeminal neuralgia, a complex        regional pain syndrome [e.g. complex regional pain syndrome I        and/or complex regional pain syndrome II], allodynia or        fibromyalgia);    -   j. respiratory pain (such as pain associated with asthma, airway        hyper-reactivity in asthma, chronic cough, e.g. in asthma and/or        chronic obstructive pulmonary disorder); or    -   k. other pain (such as pain associated with hormonal therapy,        diabetes, hypothyroidism, epilepsy, ataxia, periodic paralysis,        acute itch or chronic itch).

In an example, the NAV-mediated disease or condition is selected frompainful diabetic neuropathy, post-herpetic neuropathy, trigeminalneuralgia, osteoarthritis, chronic back pain, nerve compression pain(e.g. sciatic nerve compression) or cancer pain; or is selected frommigraine, post-operative pain and fibromyalgia.

In an example, the ligand of the invention comprises an anti-huma Nav1.7binding site, wherein the binding site is a human or humanized bindingsite, eg, the binding site comprises or consists of a human or humanizedantibody variable domain or plurality of variable domains (eg, humanVH/VL binding site(s)). Additionally or alternatively, the ligandcomprises one or more human antibody constant regions (eg, a humanantibody CH1, CH2, CH3 (or all of these) or Fc). In an example, theligand is an antibody that comprises human or humanized variable regionsand human constant regions (eg, bearing one or more mutations to enhanceor dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) fortreating or reducing the risk of a Nav1.7-mediated disease or conditionin a human in need thereof, the method comprising administering to saidhuman said ligand, wherein the ligand specifically binds a huma Nav1.7protein that comprises an amino acid selected from the group consistingof 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an aminoacid other than R), 328X (wherein X is an amino acid other than Y),395K, 459X (wherein X is an amino acid other than S), 693 X (wherein Xis an amino acid other than E), 767X (wherein X is an amino acid otherthan I), 830X (wherein X is an amino acid other than R), 897X (wherein Xis an amino acid other than W), 1200L, 1235L, 1488X (wherein X is anamino acid other than R), 1659X (wherein X is an amino acid other thanK), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G. Said human comprises a nucleotide sequenceencoding said Nav1.7 protein comprising said amino acid selected fromthe group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693 X (wherein X is an amino acid other than E), 767X (wherein X isan amino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the invention provides a method of targeting Nav1.7 in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a huma Nav1.7protein that comprises an amino acid selected from the group consistingof 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an aminoacid other than R), 328X (wherein X is an amino acid other than Y),395K, 459X (wherein X is an amino acid other than S), 693 X (wherein Xis an amino acid other than E), 767X (wherein X is an amino acid otherthan I), 830X (wherein X is an amino acid other than R), 897X (wherein Xis an amino acid other than W), 1200L, 1235L, 1488X (wherein X is anamino acid other than R), 1659X (wherein X is an amino acid other thanK), 1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,1002L, 1161W and 1919G. Said human comprises a nucleotide sequenceencoding said Nav1.7 protein comprising said amino acid selected fromthe group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X(wherein X is an amino acid other than R), 328X (wherein X is an aminoacid other than Y), 395K, 459X (wherein X is an amino acid other thanS), 693 X (wherein X is an amino acid other than E), 767X (wherein X isan amino acid other than I), 830X (wherein X is an amino acid other thanR), 897X (wherein X is an amino acid other than W), 1200L, 1235L, 1488X(wherein X is an amino acid other than R), 1659X (wherein X is an aminoacid other than K), 1689X (wherein X is an amino acid other than W),422D, 490N, 943L, 1002L, 1161W and 1919G. In an example, the human issuffering from or at risk of a Nav1.7-mediated disease or condition. Inan example, the method treats or reduces the risk of a Nav1.7-mediateddisease or condition in the human.

An example also provides a ligand (eg, an antibody or antibody fragment)for targeting Nav1.7 in a human, the method comprising administering tosaid human said ligand, wherein the ligand specifically binds a humaNav1.7 protein that comprises an amino acid selected from the groupconsisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein Xis an amino acid other than R), 328X (wherein X is an amino acid otherthan Y), 395K, 459X (wherein X is an amino acid other than S), 693 X(wherein X is an amino acid other than E), 767X (wherein X is an aminoacid other than I), 830X (wherein X is an amino acid other than R), 897X(wherein X is an amino acid other than W), 1200L, 1235L, 1488X (whereinX is an amino acid other than R), 1659X (wherein X is an amino acidother than K), 1689X (wherein X is an amino acid other than W), 422D,490N, 943L, 1002L, 1161W and 1919G. Said human comprises a nucleotidesequence encoding said Nav1.7 protein comprising said amino acidselected from the group consisting of 136I, 216S, 241T, 395K, 848T,858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,1627K, 277X (wherein X is an amino acid other than R), 328X (wherein Xis an amino acid other than Y), 395K, 459X (wherein X is an amino acidother than S), 693 X (wherein X is an amino acid other than E), 767X(wherein X is an amino acid other than I), 830X (wherein X is an aminoacid other than R), 897X (wherein X is an amino acid other than W),1200L, 1235L, 1488X (wherein X is an amino acid other than R), 1659X(wherein X is an amino acid other than K), 1689X (wherein X is an aminoacid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. In anexample, the human is suffering from or at risk of a Nav1.7-mediateddisease or condition. In an example, the method treats or reduces therisk of a Nav1.7-mediated disease or condition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40; andwherein (ii) said human comprises a nucleotide sequence encoding saidNav1.7 protein comprising said amino acid selected from the groupconsisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein Xis an amino acid other than R), 328X (wherein X is an amino acid otherthan Y), 395K, 459X (wherein X is an amino acid other than S), 693 X(wherein X is an amino acid other than E), 767X (wherein X is an aminoacid other than I), 830X (wherein X is an amino acid other than R), 897X(wherein X is an amino acid other than W), 1200L, 1235L, 1488X (whereinX is an amino acid other than R), 1659X (wherein X is an amino acidother than K), 1689X (wherein X is an amino acid other than W), 422D,490N, 943L, 1002L, 1161W and 1919G.

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises a nucleotide sequence encoding said Nav1.7 protein comprisingsaid amino acid selected from the group consisting of 136I, 216S, 241T,395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T,1462V, 1464I, 1627K, 277X (wherein X is an amino acid other than R),328X (wherein X is an amino acid other than Y), 395K, 459X (wherein X isan amino acid other than S), 693 X (wherein X is an amino acid otherthan E), 767X (wherein X is an amino acid other than I), 830X (wherein Xis an amino acid other than R), 897X (wherein X is an amino acid otherthan W), 1200L, 1235L, 1488X (wherein X is an amino acid other than R),1659X (wherein X is an amino acid other than K), 1689X (wherein X is anamino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.

In an example, the ligand, antibody or fragment is for treating orreducing the risk of (or treats or reduces the risk of) pain or itching,optionally wherein the antibody is a humanized mouse or Camelid (eg,llama or camel) antibody.

In a specific embodiment, the anti-Nav1.7 ligand, antibody or fragmentalso specifically binds to another Nav selected from Nav1.1-1.9, eg, itspecifically binds to Nav1.8 or 1.9.

In a specific embodiment, the anti-Nav1.7 ligand, antibody or fragmentof present invention comprises an Fc region, wherein the Fc regioncomprises at least one non-native amino acid residue selected from thegroup consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341,234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H,235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H,239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241R. 243W, 243L243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L,256E, 256M, 262I, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 264I,264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F,265V, 2651, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E,269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D,296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981,298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F,316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W,327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T,328H, 328A, 329F, 329H, 329Q, 330K, 330G, 3301, 330C, 330L, 330Y, 330V,3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q,331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331I, 332D,332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E,370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered bythe EU index as set forth in Kabat. Optionally, the Fc region maycomprise additional and/or alternative non-native amino acid residuesknown to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919;WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention is fortreating or preventing or reducing the risk of (or treats or prevents orreduces the risk of), for example, any disease or condition disclosed inWO2011/051351, the disclosure of which diseases and conditions areincorporated herein by reference for potential inclusion in one or moreclaims herein. Guidance on obtaining and testing antibodies can also befound in that PCT application.

Further encompassed by the invention is the use of the ligand, antibodyor fragment in the manufacture of a medicament for use to attenuate orinhibit a Nav1.7-mediated disease or disorder in a human.Nav1.7-mediated or related disorders which are treated by the ligand,antibody or fragment of the invention include, for example, a pain oritching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving Nav1.7expression and/or activity. One embodiment, among others, is a method oftreatment comprising administering an effective amount of a ligand,antibody or fragment of the invention to a patient in need thereof,wherein functional consequences of Nav1.7 activation are decreased.Another embodiment, among others, is a method of treatment comprising(i) identifying a patient demonstrating Nav1.7 expression or activity,and (ii) administering an effective amount of a ligand, antibody orfragment of the invention to the patient, wherein a functionalconsequence of Nav1.7 activation are attenuated. An effective amountaccording to the invention is an amount that modulates (e.g. decreases)the functional consequences of Nav1.7 activation so as to modulate (e.g.decrease or lessen) the severity of at least one symptom of theparticular disease or disorder being treated, but not necessarily curethe disease or disorder. Accordingly, one embodiment of the invention isa method of treating or reducing the severity of at least one symptom ofany of the disorders referred to herein, comprising administering to apatient in need thereof an effective amount of one or more ligands,antibodies or fragments of the present invention alone or in a combinedtherapeutic regimen with another appropriate medicament known in the artor described herein such that the severity of at least one symptom ofany of the disorders is reduced. Another embodiment of the invention,among others, is a method of antagonizing at least one effect of Nav1.7comprising contacting with or administering an effective amount of oneor more ligands, antibodies or fragments of the present invention suchthat said at least one effect of Nav1.7 is antagonized, e.g. the abilityof Nav1.7 to form an ion channel, such as a sodium channel

Tailoring Antibodies to Rarer Nav1.7 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1),the invention includes the possibility to tailor treatment of humansfurther by selecting antibody-based ligands with variable domains and/orconstant domains based on gene segments found in many humans of theethnic populations where the variant TOI forms are found to meet theselection criteria of the invention. This also applies mutatis mutandiswhere the TOI is huma Nav1.7, as in the present example. Thus, alldisclosure herein relating to tailoring variable and/or constant domainsapply to the present example, relating to Nav1.7 and is combinable foruse in one or more claims herein.

As described in Example 1, an example is provided for ligands comprisingantibody VH domains derived from recombination of human IGHV genesegments comprising selected nucleotides at positions in the HCDR1 orFW3 where there is variability in humans (ie, where SNPs occur inhumans).

Further information is provided in Table 4, which shows variation atthese positions, as well as the variant distributions across the 1000Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ, or constant region gene segments (see further Table 9 forrepresentative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4. (vi)wherein the ligand comprises a human gamma-2 heavy chain constant regionthat comprises an amino acid selected from the group consisting of a Proat position 72 shown in SEQ ID NO: 6, an Asn at position 75 shown in SEQID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Val at position161 shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID NO:6 and wherein said human comprises (i) an IGHG2*01 human heavy chainconstant region gene segment, or the human expresses antibodiescomprising human gamma-2 heavy chain constant regions comprising saidselected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val atposition 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQID NO: 6.(vii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.

-   (viii) wherein the ligand comprises a human IGLC1*01 lambda chain    constant region and wherein said human comprises (i) a human    IGLC1*01 lambda chain constant region gene segment, or the human    expresses antibodies comprising human IGLC1*01 lambda chain constant    regions.-   (ix) wherein the ligand comprises a human gamma-4 heavy chain    constant region that comprises a Leu at position 189 shown in SEQ ID    NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein    said human comprises (i) an IGHG4*01 human heavy chain constant    region gene segment, or the human expresses antibodies comprising    human gamma-4 heavy chain constant regions comprising a Leu at    position 189 shown in SEQ ID NO: 73 or an Arg at position 289 shown    in SEQ ID NO: 73.-   (x) wherein the ligand comprises a human gamma-3 heavy chain    constant region encoded by a first human IGHG3 (eg, IGHG3*01)    constant region gene segment and wherein said human comprises (i)    said first constant region gene segment (eg, an IGHG3*01), or the    human expresses antibodies comprising human gamma-3 heavy chain    constant regions encoded by said first human IGHG3 (eg, IGHG3*01)    constant region gene segment.-   (xi) wherein the ligand comprises a human epsilon heavy chain    constant region encoded by a first human epsilon heavy chain    constant region gene segment and wherein said human comprises (i)    said first constant region gene segment, or the human expresses    antibodies comprising human epsilon heavy chain constant regions    encoded by said first constant region gene segment.-   (xii) wherein the ligand comprises a human mu heavy chain constant    region encoded by a first human mu heavy chain constant region gene    segment and wherein said human comprises (i) said first constant    region gene segment, or the human expresses antibodies comprising    human mu heavy chain constant regions encoded by said first constant    region gene segment.-   (xiii) wherein the ligand comprises a human alpha heavy chain    constant region encoded by a first human alpha heavy chain constant    region gene segment and wherein said human comprises (i) said first    constant region gene segment, or the human expresses antibodies    comprising human alpha heavy chain constant regions encoded by said    first constant region gene segment.-   (xiv) wherein the ligand comprises a human delta heavy chain    constant region encoded by a first human delta heavy chain constant    region gene segment and wherein said human comprises (i) said first    constant region gene segment, or the human expresses antibodies    comprising human delta heavy chain constant regions encoded by said    first constant region gene segment.-   (xv) wherein the ligand comprises a human kappa light chain constant    region encoded by a first human kappa light chain constant region    gene segment and wherein said human comprises (i) said first    constant region gene segment, or the human expresses antibodies    comprising human kappa light chain constant regions encoded by said    first constant region gene segment.-   (xvi) wherein the ligand comprises a human lambda light chain    constant region encoded by a first human lambda light chain constant    region gene segment and wherein said human comprises (i) said first    constant region gene segment, or the human expresses antibodies    comprising human lambda light chain constant regions encoded by said    first constant region gene segment.

These examples, although written in the context of the present Nav1.7example, are applicable to the invention as it relates to any other TOIherein and thus can be combined and used in claims herein relating toany TOI. In any of these example, the ligand is optionally an antibody,eg, a human or humanised antibody (eg, a humanised mouse, rat or Camelidantibody).

For ADCC efficacy for human constant regions is: IgG1≧IgG3>>IgG4≧IgG2

For CDC efficacy for human constant regions is: IgG3≧IgG1>>IgG2≈IgG4

Thus, it may be advantageous for Nav1.7 ligands, for the ligand tocomprise a gamma-4 or gamma-2 constant region (eg, as per the examplesabove). For example, the gamma-4 is an IgG4PE (ie, a gamma-4 constantregion with 228Pro and 235Glu). For example the gamma-2 is an IgG2Aa.

For example, as per example (ix), the inventor identified thepossibility of addressing IGH-gamma-4 variation and identified utilityfor variations 189L and 289R individually or in combination, since theseresidues are part of the CH3 domain, and as such they form part ofantibody Fc regions. Thus, matching of these CH3 variations with thepatient is especially beneficial for reasons as discussed above. Thus,in this example the ligand of the invention comprises or consists of anantibody that comprises a human gamma-4 heavy chain constant region thatcomprises a Leu corresponding to position 189 of SEQ ID NO: 73 or an Argcorresponding to position 289 of SEQ ID NO: 73 and wherein the genome ofthe human comprises a gamma-4 heavy chain constant region nucleotidesequence that encodes such a Leu and/or Arg or the human expressesantibodies comprising human gamma-4 constant regions comprising such aLeu and/or Arg.

Determination of Specific Binding of Ligands of the Invention to NAV1.7Variants

The specific binding of ligands of the invention to Nav1.7 variants canbe performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a huma Nav1.7 protein    that comprises an amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.7 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a huma Nav1.7 protein        that comprises an amino acid selected from the group consisting        of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C,        1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X        is an amino acid other than R), 328X (wherein X is an amino acid        other than Y), 395K, 459X (wherein X is an amino acid other than        S), 693 X (wherein X is an amino acid other than E), 767X        (wherein X is an amino acid other than I), 830X (wherein X is an        amino acid other than R), 897X (wherein X is an amino acid other        than W), 1200L, 1235L, 1488X (wherein X is an amino acid other        than R), 1659X (wherein X is an amino acid other than K), 1689X        (wherein X is an amino acid other than W), 422D, 490N, 943L,        1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The method of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.7 protein comprising said amino acid selected    from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,    858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,    1627K, 277X (wherein X is an amino acid other than R), 328X (wherein    X is an amino acid other than Y), 395K, 459X (wherein X is an amino    acid other than S), 693 X (wherein X is an amino acid other than E),    767X (wherein X is an amino acid other than I), 830X (wherein X is    an amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G and/or (b) a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.7 protein comprising said amino acid selected        from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,        858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,        1464I, 1627K, 277X (wherein X is an amino acid other than R),        328X (wherein X is an amino acid other than Y), 395K, 459X        (wherein X is an amino acid other than S), 693 X (wherein X is        an amino acid other than E), 767X (wherein X is an amino acid        other than I), 830X (wherein X is an amino acid other than R),        897X (wherein X is an amino acid other than W), 1200L, 1235L,        1488X (wherein X is an amino acid other than R), 1659X (wherein        X is an amino acid other than K), 1689X (wherein X is an amino        acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs6746030, rs3750904, rs58022607, rs4369876,    rs13402180 and rs12478318.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.

Preferably herein the Nav1.7 mutation is 1161W (also known as 1150W insome publications, eg, Reimann et al (Proc Natl Acad Sci USA. 2010 Mar.16; 107(11):5148-53. doi: 10.1073/pnas.0913181107. Epub 2010 Mar. 8;“Pain perception is altered by a nucleotide polymorphism in SCN9A”;Reimann F et al). This describes the correlation of 1150W (ie, 1161W)with increased pain perception (lower threshold) in normal humans.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.7-mediated disease    or condition (eg, asthma) in a human in need thereof, wherein the    ligand specifically binds a human Nav1.7 protein that comprises an    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.7 in a human, wherein the ligand        specifically binds a huma Nav1.7 protein that comprises an amino        acid selected from the group consisting of 136I, 216S, 241T,        395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,        1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid        other than R), 328X (wherein X is an amino acid other than Y),        395K, 459X (wherein X is an amino acid other than S), 693 X        (wherein X is an amino acid other than E), 767X (wherein X is an        amino acid other than I), 830X (wherein X is an amino acid other        than R), 897X (wherein X is an amino acid other than W), 1200L,        1235L, 1488X (wherein X is an amino acid other than R), 1659X        (wherein X is an amino acid other than K), 1689X (wherein X is        an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and        1919G;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The ligand of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.7 protein comprising said amino acid selected        from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,        858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,        1464I, 1627K, 277X (wherein X is an amino acid other than R),        328X (wherein X is an amino acid other than Y), 395K, 459X        (wherein X is an amino acid other than S), 693 X (wherein X is        an amino acid other than E), 767X (wherein X is an amino acid        other than I), 830X (wherein X is an amino acid other than R),        897X (wherein X is an amino acid other than W), 1200L, 1235L,        1488X (wherein X is an amino acid other than R), 1659X (wherein        X is an amino acid other than K), 1689X (wherein X is an amino        acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs6746030, rs3750904, rs58022607, rs4369876,    rs13402180 and rs12478318.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a huma Nav1.7 protein    that comprises an amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.7 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a huma Nav1.7 protein        that comprises an amino acid selected from the group consisting        of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C,        1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X        is an amino acid other than R), 328X (wherein X is an amino acid        other than Y), 395K, 459X (wherein X is an amino acid other than        S), 693 X (wherein X is an amino acid other than E), 767X        (wherein X is an amino acid other than I), 830X (wherein X is an        amino acid other than R), 897X (wherein X is an amino acid other        than W), 1200L, 1235L, 1488X (wherein X is an amino acid other        than R), 1659X (wherein X is an amino acid other than K), 1689X        (wherein X is an amino acid other than W), 422D, 490N, 943L,        1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The method of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.7 protein comprising said amino acid selected    from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,    858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,    1627K, 277X (wherein X is an amino acid other than R), 328X (wherein    X is an amino acid other than Y), 395K, 459X (wherein X is an amino    acid other than S), 693 X (wherein X is an amino acid other than E),    767X (wherein X is an amino acid other than I), 830X (wherein X is    an amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G and/or (b) a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex    -   determines that the human comprises the Nav1.7 protein        comprising said amino acid selected from the group consisting of        136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C,        1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X        is an amino acid other than R), 328X (wherein X is an amino acid        other than Y), 395K, 459X (wherein X is an amino acid other than        S), 693 X (wherein X is an amino acid other than E), 767X        (wherein X is an amino acid other than I), 830X (wherein X is an        amino acid other than R), 897X (wherein X is an amino acid other        than W), 1200L, 1235L, 1488X (wherein X is an amino acid other        than R), 1659X (wherein X is an amino acid other than K), 1689X        (wherein X is an amino acid other than W), 422D, 490N, 943L,        1002L, 1161W and 1919G.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs6746030, rs3750904, rs58022607, rs4369876,    rs13402180 and rs12478318.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.-   22. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof.-   23. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.7-mediated disease    or condition (eg, pain) in a human in need thereof, wherein the    ligand specifically binds a huma Nav1.7 protein that comprises an    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.7 in a human, wherein the ligand        specifically binds a huma Nav1.7 protein that comprises an amino        acid selected from the group consisting of 136I, 216S, 241T,        395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,        1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid        other than R), 328X (wherein X is an amino acid other than Y),        395K, 459X (wherein X is an amino acid other than S), 693 X        (wherein X is an amino acid other than E), 767X (wherein X is an        amino acid other than I), 830X (wherein X is an amino acid other        than R), 897X (wherein X is an amino acid other than W), 1200L,        1235L, 1488X (wherein X is an amino acid other than R), 1659X        (wherein X is an amino acid other than K), 1689X (wherein X is        an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and        1919G;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and wherein (ii)        said human comprises a nucleotide sequence encoding said Nav1.7        protein comprising said amino acid selected from the group        consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,        1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K,        277X (wherein X is an amino acid other than R), 328X (wherein X        is an amino acid other than Y), 395K, 459X (wherein X is an        amino acid other than S), 693 X (wherein X is an amino acid        other than E), 767X (wherein X is an amino acid other than I),        830X (wherein X is an amino acid other than R), 897X (wherein X        is an amino acid other than W), 1200L, 1235L, 1488X (wherein X        is an amino acid other than R), 1659X (wherein X is an amino        acid other than K), 1689X (wherein X is an amino acid other than        W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The ligand of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.7 protein comprising said amino acid selected        from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,        858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,        1464I, 1627K, 277X (wherein X is an amino acid other than R),        328X (wherein X is an amino acid other than Y), 395K, 459X        (wherein X is an amino acid other than S), 693 X (wherein X is        an amino acid other than E), 767X (wherein X is an amino acid        other than I), 830X (wherein X is an amino acid other than R),        897X (wherein X is an amino acid other than W), 1200L, 1235L,        1488X (wherein X is an amino acid other than R), 1659X (wherein        X is an amino acid other than K), 1689X (wherein X is an amino        acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs6746030, rs3750904, rs58022607, rs4369876,    rs13402180 and rs12478318.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.-   22. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof.-   23. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.7-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a huma Nav1.7 protein    that comprises an amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.7 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a huma Nav1.7 protein        that comprises an amino acid selected from the group consisting        of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C,        1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X        is an amino acid other than R), 328X (wherein X is an amino acid        other than Y), 395K, 459X (wherein X is an amino acid other than        S), 693 X (wherein X is an amino acid other than E), 767X        (wherein X is an amino acid other than I), 830X (wherein X is an        amino acid other than R), 897X (wherein X is an amino acid other        than W), 1200L, 1235L, 1488X (wherein X is an amino acid other        than R), 1659X (wherein X is an amino acid other than K), 1689X        (wherein X is an amino acid other than W), 422D, 490N, 943L,        1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The method of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.7 protein comprising said amino acid selected    from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,    858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,    1627K, 277X (wherein X is an amino acid other than R), 328X (wherein    X is an amino acid other than Y), 395K, 459X (wherein X is an amino    acid other than S), 693 X (wherein X is an amino acid other than E),    767X (wherein X is an amino acid other than I), 830X (wherein X is    an amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G and/or (b) a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G, optionally, wherein the determining step is    performed before administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.7 protein comprising said amino acid selected        from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,        858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,        1464I, 1627K, 277X (wherein X is an amino acid other than R),        328X (wherein X is an amino acid other than Y), 395K, 459X        (wherein X is an amino acid other than S), 693 X (wherein X is        an amino acid other than E), 767X (wherein X is an amino acid        other than I), 830X (wherein X is an amino acid other than R),        897X (wherein X is an amino acid other than W), 1200L, 1235L,        1488X (wherein X is an amino acid other than R), 1659X (wherein        X is an amino acid other than K), 1689X (wherein X is an amino        acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs6746030, rs3750904, rs58022607, rs4369876,    rs13402180 and rs12478318.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.-   22. The method of any preceding clause, wherein the human gamma-2    heavy chain constant region of the ligand comprises IGHG2*01 amino    acid sequence or an ADCC inactivated version thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.7-mediated disease    or condition (eg, pain) in a human in need thereof, wherein the    ligand specifically binds a huma Nav1.7 protein that comprises an    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G;    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.7 in a human, wherein the ligand        specifically binds a huma Nav1.7 protein that comprises an amino        acid selected from the group consisting of 136I, 216S, 241T,        395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,        1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid        other than R), 328X (wherein X is an amino acid other than Y),        395K, 459X (wherein X is an amino acid other than S), 693 X        (wherein X is an amino acid other than E), 767X (wherein X is an        amino acid other than I), 830X (wherein X is an amino acid other        than R), 897X (wherein X is an amino acid other than W), 1200L,        1235L, 1488X (wherein X is an amino acid other than R), 1659X        (wherein X is an amino acid other than K), 1689X (wherein X is        an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and        1919G;    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.    -   In an example, the human is suffering from or at risk of a        Nav1.7-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.7-mediated disease or condition in the human.-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of any one of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P and 1449V; optionally wherein said    disease or condition is a pain disease or condition.-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I and 1627K; optionally wherein said disease or    condition is a pain disease or condition.-   4. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 277X (wherein X is an amino    acid other than R), 328X (wherein X is an amino acid other than Y),    395K, 459X (wherein X is an amino acid other than S), 693 X (wherein    X is an amino acid other than E), 767X (wherein X is an amino acid    other than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K) and 1689X (wherein X is an amino acid other    than W); optionally wherein said disease or condition is a pain    disease or condition.-   5. The ligand of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.7    protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G    and/or a Nav1.7 protein comprising said amino acid selected from the    group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P,    1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X    (wherein X is an amino acid other than R), 328X (wherein X is an    amino acid other than Y), 395K, 459X (wherein X is an amino acid    other than S), 693 X (wherein X is an amino acid other than E), 767X    (wherein X is an amino acid other than I), 830X (wherein X is an    amino acid other than R), 897X (wherein X is an amino acid other    than W), 1200L, 1235L, 1488X (wherein X is an amino acid other than    R), 1659X (wherein X is an amino acid other than K), 1689X (wherein    X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and    1919G.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G and/or (b) a Nav1.7 protein comprising said    amino acid selected from the group consisting of 136I, 216S, 241T,    395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F,    1461T, 1462V, 1464I, 1627K, 277X (wherein X is an amino acid other    than R), 328X (wherein X is an amino acid other than Y), 395K, 459X    (wherein X is an amino acid other than S), 693 X (wherein X is an    amino acid other than E), 767X (wherein X is an amino acid other    than I), 830X (wherein X is an amino acid other than R), 897X    (wherein X is an amino acid other than W), 1200L, 1235L, 1488X    (wherein X is an amino acid other than R), 1659X (wherein X is an    amino acid other than K), 1689X (wherein X is an amino acid other    than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, optionally,    wherein the determining step is performed before administration of    the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.7 protein comprising said amino acid    selected from the group consisting of 136I, 216S, 241T, 395K, 848T,    858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,    1464I, 1627K, 277X (wherein X is an amino acid other than R), 328X    (wherein X is an amino acid other than Y), 395K, 459X (wherein X is    an amino acid other than S), 693 X (wherein X is an amino acid other    than E), 767X (wherein X is an amino acid other than I), 830X    (wherein X is an amino acid other than R), 897X (wherein X is an    amino acid other than W), 1200L, 1235L, 1488X (wherein X is an amino    acid other than R), 1659X (wherein X is an amino acid other than K),    1689X (wherein X is an amino acid other than W), 422D, 490N, 943L,    1002L, 1161W and 1919G.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G or        comprising an antisense sequence of said contiguous nucleotides,        wherein said sequence of contiguous nucleotides comprises a        nucleotide sequence encoding said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, thereby        forming a complex when at least one nucleotide sequence encoding        the Nav1.7 protein comprising said amino acid selected from the        group consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F,        863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I,        1627K, 277X (wherein X is an amino acid other than R), 328X        (wherein X is an amino acid other than Y), 395K, 459X (wherein X        is an amino acid other than S), 693 X (wherein X is an amino        acid other than E), 767X (wherein X is an amino acid other than        I), 830X (wherein X is an amino acid other than R), 897X        (wherein X is an amino acid other than W), 1200L, 1235L, 1488X        (wherein X is an amino acid other than R), 1659X (wherein X is        an amino acid other than K), 1689X (wherein X is an amino acid        other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G is        present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.7 protein comprising said amino acid selected        from the group consisting of 136I, 216S, 241T, 395K, 848T, 858H,        858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V,        1464I, 1627K, 277X (wherein X is an amino acid other than R),        328X (wherein X is an amino acid other than Y), 395K, 459X        (wherein X is an amino acid other than S), 693 X (wherein X is        an amino acid other than E), 767X (wherein X is an amino acid        other than I), 830X (wherein X is an amino acid other than R),        897X (wherein X is an amino acid other than W), 1200L, 1235L,        1488X (wherein X is an amino acid other than R), 1659X (wherein        X is an amino acid other than K), 1689X (wherein X is an amino        acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.7 protein comprising said amino acid selected from the group    consisting of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V,    996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein    X is an amino acid other than R), 328X (wherein X is an amino acid    other than Y), 395K, 459X (wherein X is an amino acid other than S),    693 X (wherein X is an amino acid other than E), 767X (wherein X is    an amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G,    optionally, wherein said human is further indicated as comprising    the nucleotide sequence of SEQ ID NO: 76, or said human is indicated    as homozygous for a nucleotide sequence encoding the Nav1.7 protein    comprising said amino acid selected from the group consisting of    136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F,    1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (wherein X is an    amino acid other than R), 328X (wherein X is an amino acid other    than Y), 395K, 459X (wherein X is an amino acid other than S), 693 X    (wherein X is an amino acid other than E), 767X (wherein X is an    amino acid other than I), 830X (wherein X is an amino acid other    than R), 897X (wherein X is an amino acid other than W), 1200L,    1235L, 1488X (wherein X is an amino acid other than R), 1659X    (wherein X is an amino acid other than K), 1689X (wherein X is an    amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is a channelopathy or associated with a channelopathy; or    is selected from the group consisting of primary erythermalgia (PE),    paroxysmal extreme pain disorder (PEPD) and channelopathy-associated    insensitivity to pain (CIP).-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more

SNPs selected from the group consisting of rs6746030, rs3750904,rs58022607, rs4369876, rs13402180 and rs12478318.

-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.-   22. The ligand of any preceding clause, wherein the human gamma-2    heavy chain constant region of the ligand comprises IGHG2*01 amino    acid sequence or an ADCC inactivated version thereof.

TABLE 4 1000 GENOMES PROJECT HUMAN POPULATIONS Below is a summary of theethnic populations as per the 1000 Genomes Project sequences. PopulationEuropean ancestry Utah residents (CEPH) with Northern and WesternEuropean ancestry (CEU) Toscani in Italia (TSI) British from England andScotland (GBR) Finnish from Finland (FIN) Iberian populations in Spain(IBS) East Asian ancestry Han Chinese in Beijing China (CHB) Japanese inToyko, Japan (JPT) Han Chinese South (CHS) Chinese Dai in Xishuangbanna(CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese in Denver,Colorado (CHD) (pilot 3 only) West African ancestry Yoruba in Ibadan,Nigeria (YRI) Luhya in Webuy, Kenya (LWK) Gambian in Western Division,The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) West AfricanPopulation (TBD) Americas African Ancestry in Southwest US (ASW) AfricanAmerican in Jackson, MS (AJM) African Caribbean in Barbados (ACB)Mexican Ancestry in Los Angeles CA (MXL) Puerto Rican in Puerto Rico(PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru (PEL)South Asian ancestry Ahom in the State of Assam, India Kayadtha inCalcutta, India Reddy in Hyderabad, India Maratha in Bombay, IndiaPunjabi in Lahore, Pakistan

TABLE 5 TOIs & Related Diseases, Conditions and Example Ligands ExampleDisease or Human TOI Condition Example Ligand Il-17a InflammatoryDisease AIN457 Uveitis Ixekizumab Rheumatoid Arthritis PsoriasisAngiotensin II Receptor Type 1 (AT₁) Hypertension LCZ696 NeprilysinHypertension LCZ696 Metabotropic Glutamate Fragile X Syndrome AFQ056Receptor 5 (Mglur5) Mavoglurant A Histone Deacetylase Cancer, Eg,Multiple LBH589 Myeloma Panobinostat Diacylglycerol acyltransferase-1Familial LCQ908 (DGAT-1) Chylomicronaemia Pradigastat SyndromeSmoothened (Smo) Basal cell carcinoma LDE225 Solid tumour MyelofibrosisMedulloblastoma Solid tumour Smoothened (Smo) receptor Basal cellcarcinoma LEQ506 Solid tumors ALK Non small cell lung LDK378 carcinoma(NSCLC) phosphatidylinositol-3-kinase (PI3K) Cancer, eg, solid tumour,BKM120 mTOR breast cancer, Renal cell AKT carcinoma (RCC), Endometrialcancer, Non- small cell lung cancer (NSCLC), prostate cancer,Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis mTOR Cancer, eg,solid tumour, BEZ235 PI3K breast cancer, Renal cell AKT carcinoma (RCC),Endometrial cancer, Non- small cell lung cancer (NSCLC), prostatecancer, Glioblastoma multiforme (GBM) CRPC GIST Myelofibrosis PI3KαCancer, eg, solid tumour, BYL719 mTOR breast cancer, Renal cell PI3Kcarcinoma (RCC), AKT Endometrial cancer, Non- small cell lung cancer(NSCLC), prostate cancer, Glioblastoma multiforme (GBM) CRPC GISTMyelofibrosis Mitogen-activated ERK kinase 1 (MEK1) Cancer, eg, solidtumour, MEK162 Mitogen-activated ERK kinase 2 (MEK2) melanoma,pancreatic, colon, lung or thyroid cancer Metastasis protein kinaseAcute myeloid leukemia PKC412 protein kinase C (AML) Midostaurin FLT-3Myelodysplastic syndrome c-KIT (MDS) Aggressive systemic mastocytosis(ASM) ActRIIB Sporadic inclusion body BYM338 myositis (sIBM) bimagrumabCD19 Cancer, eg, CTL019 (formerly CART- Leukaemia, mast 19) cellleukemia, Acute Lymphoblastic Leukemia, Chronic lymphocytic leukemia(CLL) or Hematological tumors 11β-hydroxylase Cushing's syndrome LCI699BRAF Cancer, eg, melanoma LGX818 Receptor Tyrosine Kinase Cancer, eg,solid TKI258 (formerly CHIR- FGFR tumour 258) Breast Cancer DovitinibEndometrial cancer Hepatocellular carcinoma Renal cell carcinoma (RCC)Bladder cancer multiple myeloma (MM), hepatocellular carcinomaendometrial cancer DAC enzyme hematologic malignancy LBH589 Multiplemyeloma panobinostat Myel\odysplastic syndrome (MDS) Myelofibrosis HSP90Cancer, eg, Breast AUY922 Cancer, gastric cancer or Non- small cell lungcancer (NSCLC) FGFR Cancer, eg, solid BGJ398 tumour Breast CancerEndometrial cancer Hepatocellular carcinoma Renal cell carcinoma (RCC)Bladder cancer multiple myeloma (MM), hepatocellular carcinomaendometrial cancer cIAP1 Cancer, eg, solid LCL161 cIAP2 tumour BreastCancer Endometrial cancer Hepatocellular carcinoma Renal cell carcinoma(RCC) Bladder cancer multiple myeloma (MM), hepatocellular carcinomaendometrial cancer Akt Cancer, eg, Solid GDC-0068 tumour, gastric RG7440cancer (eg, castration-resistant prostate cancer), prostate cancer,gastroesophageal junction cancer CD22 Hematologic malignancies DCDT2980SNon-Hodgkin's lymphoma RG7593 (eg, relapsed or refractory follicularnon-Hodgkin's lymphoma) Diffuse large B-cell lymphoma (eg, relapsed orrefractory diffuse large B- cell lymphoma) CD79b Hematologicmalignancies DCDS4501A Non-Hodgkin's lymphoma RG7596 (eg, relapsed orrefractory follicular non-Hodgkin's lymphoma) Diffuse large B-celllymphoma (eg, relapsed or refractory diffuse large B- cell lymphoma)endothelin B receptor (ETBR) Melanoma, eg, metastatic DEDN6526A orunresectable melanoma RG7636 HER3 Cancer, eg, metastatic MEHD7945Aepithelial tumour, RG7597 metastatic squamous cell carcinoma of the headand neck cancer, metastatic colorectal cancer EGFR Cancer, eg,metastatic MEHD7945A epithelial tumour, RG7597 metastatic squamous cellNecitumumab carcinoma of the head and neck cancer, metastatic colorectalcancer MUC16 Cancer, eg, ovarian cancer DMUC5754A (eg,platinum-resistant RG7458 ovarian cancer) sodium-dependent phosphateCancer, eg, metastatic non- DNIB0600A transport protein 2b (NaPi2b)squamous non-small cell RG7599 lung cancer, ovarian cancer (eg,platinum-resistant ovarian cancer) PDL1 (programmed death ligand 1)Cancer, eg, solid tumour MPDL3280A metastatic melanoma RG7446 non-smallcell lung cancer STEAP1 (six-transmembrane epithelial Cancer, eg,prostate cancer DSTP3086S antigen of the prostate 1) (eg, metastaticcastration- RG7450 resistant prostate cancer) Bcl-2 Cancer, eg,leukaemia (eg, GDC-0199 chronic lymphocytic RG7601 leukaemia),non-Hodgkin's lymphoma Checkpoint kinase 1 (ChK1) Cancer, eg, solidtumour, GDC-0425 refractory solid tumour or RG7602 lymphoma GDC-0575RG7741 mitogen activated protein kinase kinase Cancer, eg, solid tumour,GDC-0973 (MAPKK) melanoma (eg, metastatic RG7421 MEK melanoma)Cobimetinib GDC-0623 RG7420 epidermal growth factor domain-like-7Cancer, eg, colorectal Parsatuzumab (EGFL7) cancer (eg, metastaticMEGF0444A colorectal cancer), NSCLC RG7414 phosphatidylinositol-3-kinase(PI3K) Cancer, eg, prostate cancer, GDC-0032 renal cell carcinoma,RG7604 endometrial cancer, breast GDC-0084 cancer (eg, HER2-negativeRG7666 metastatic breast cancer, Pictilisib metastatic hormone GDC-0941receptor-positive breast RG7321 cancer), solid tumour mTOR Cancer, eg,prostate cancer, DC-0980 TORC1 renal cell carcinoma, RG7422 TORC2endometrial cancer, breast PI3K cancer (eg, HER2-negative metastaticbreast cancer, metastatic hormone receptor-positive breast cancer),solid tumour IL17 Autoimmune disease Secukinumab Ankylosing spondylitis;ixekizumab Asthma; Multiple myeloma; Multiple sclerosis; Polymyalgiarheumatica; Psoriasis; Psoriatic arthritis; Rheumatoid arthritis;Uveitis M1 prime segment of membrane IgE Allergic Asthma QuilizumabMEMP1972A RG7449 IFN alpha Autoimmune disease Rontalizumab Systemiclupus erythematosus PCSK9 (proprotein convertase coronary heart diseaseMPSK3169A subtilisin/kexin type 9) (CHD) or high risk of CHD RG7652hyperlipidaemia hypercholesterolaemia stroke atherosclerosis acardiovascular disease or condition a condition associated with elevatedLDL Vascular Endothelial Growth Factor-A Diabetic Macular Edema EYLEA  ®(VEGF-A) (DME), Aflibercept Placental Growth Factor (PlGF) BranchRetinal Vein AVASTIN  ™ Occlusion (BRVO) LUCENTIS  ™ Wet age-relatedmacular degeneration (Wet AMD) IL-6 receptor Inflammatory disease, eg,Sarilumab rheumatoid arthritis REGN88 Uveitis (eg, non-infectiousuveitis) Ankylosing spondylitis; Cancer PCSK9 (proprotein convertasecoronary heart disease Alirocumab subtilisin/kexin type 9) (CHD) or highrisk of CHD REGN727 hyperlipidaemia LY3015014 hypercholesterolaemiastroke atherosclerosis a cardiovascular disease or condition a conditionassociated with elevated LDL NGF Osteoarthritis Fasinumab Pain REGN475IL-4 receptor alpha Allergic asthma Dupilumab IL-13 receptoreosinophilic asthma REGN668 Atopic dermatitis delta-like ligand-4 (Dll4)Cancer Enoticumab REGN421 angiopoietin-2 (Ang2) Cancer NesvacumabREGN910 GDF8 Metabolic disorder REGN1033 cancer, obesity, diabetes,LY2495655 arthritis, multiple sclerosis, muscular dystrophy, amyotrophiclateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis,osteopenia, metabolic syndromes (including, but not limited to diabetes,obesity, nutritional disorders, organ atrophy, chronic obstructivepulmonary disease and anorexia) Disuse muscle atrophy cancer-relatedcachexia ERBB3 Cancer REGN1400 angiopoietin-1 (Ang1) Cancer, eg, ovariancancer AMG386 angiopoietin-1 (Ang2) VEGF receptor Cancer Motesanib PDGFreceptor NSCLC AMG 706 Stem cell factor receptor Breast cancer Thyroidcancer type 1 insulin-like growth factor Cancer Ganitumab receptor(IGF1R) Breast cancer Linsitinib Pancreatic cancer ASP7487 hepatocytegrowth factor (HGF) Cancer Rilotumumab Breast cancer Pancreatic cancerHER3 Cancer AMG 888 ErbB3 Breast cancer U3-1287 Pancreatic cancer IL-17receptor Inflammatory disease AMG 827 Asthma brodalumab Psoriasissclerostin Bone-related condition AMG 785 postmenopausal CDP7851osteoporosis fracture healing glucokinase Diabetes AMG 151 ARRY-403PCSK9 (proprotein convertase coronary heart disease AMG 145subtilisin/kexin type 9) (CHD) or high risk of CHD Evolocumabhyperlipidaemia hypercholesterolaemia stroke atherosclerosis acardiovascular disease or condition a condition associated with elevatedLDL VLA 2 Inflammatory bowel SAR339658 Integrin α2β1 disease IL-4Idiopathic pulmonary SAR156597 IL-13 fibrosis lysophosphatidic acidreceptor Systemic sclerosis SAR100842 LPA-1 fibrosis LPA-3 Androgenreceptor Cancer, eg, prostate cancer, MDV3100 breast cancer enzalutamideHER1 Cancer, eg, NSCLC Erlotinib EGFR ERBITUX  ™ VECTIBIX  ™ VEGFreceptor 1 Cancer, eg, colorectal ASP4130 VEGF receptor 2 cancer, breastcancer tivozanib VEGF receptor 3 JAK Inflammatory disease, eg, ASP015KJAK1 rheumatoid arthritis Baricitinib JAK2 Diabetic nephropathy CD40Prevention of organ ASP1240 transplant rejection GnRH EndometriosisASP1707 Cancer, eg, prostate cancer degarelix PDE9 Lower unirary tractASP4901 symptoms associated with benign prostatic hyperplasia TNF alphaInflammatory disease, eg, Certolizumab pegol rheumatoid arthritis,psoriasis, chrohn's disease, IBD Programmed cell death protein 1 CancerNivolumab Chronic myelocytic MK-3475 leukemia; Hepatitis C virusinfection; Hepatocellular carcinoma; Hodgkins disease; Melanoma;Multiple myeloma; Non- Hodgkin lymphoma; Non- small-cell lung cancer;Renal cell carcinoma; Solid tumor; Stage IV melanoma Hepatocyte growthfactor Cancer onartuzumab MET Glioblastoma; Hepatocellular carcinoma;Metastatic colorectal cancer; Metastatic non small cell lung cancer;Metastatic stomach cancer; Non-small-cell lung cancer Angiopoietinligand-1 Breast tumor; Cancer; trebananib Angiopoietin ligand-2Colorectal tumor; Fallopian Tek tyrosine kinase receptor tube cancer;Gastrointestinal tumor; Glioblastoma; Hepatocellular carcinoma;Metastatic esophageal cancer; Metastatic gastrointestinal cancer;Metastatic non small cell lung cancer; Metastatic ovary cancer;Metastatic renal cancer; Ovary tumor; Peritoneal tumor; Transitionalcell carcinoma CD37 modulator Cancer elotuzumab Lymphocyte functionantigen-3 receptor Multiple myeloma daclizumab SLAM family member 7Multiple sclerosis IL-2 IL-2 receptor alpha EGFR Cancer necitumumabMetastatic non small cell lung cancer; Solid tumor IL-5 Asthma;Eosinophilic reslizumab esophagitis B-lymphocyte cell adhesion moleculeCancer, eg Acute Inotuzumab CD22 lymphoblastic leukemia; inotuzumabozogamicin Follicle center lymphoma; epratuzumab Non-Hodgkin lymphoma;moxetumomab Systemic lupus moxetumomab pasudotox erythematosus, hairycell leukaemia IL1 beta Acne vulgaris; gevokizumab Atherosclerosis;Behcets disease; Cardiovascular disease; Dermatomyositis; Insulindependent diabetes; Multiple myeloma; Osteoarthritis; Paraproteinemia;Polymyositis; Pyoderma gangrenosum; Scleritis; Uveitis CD20 CancerOcrelizumab Multiple sclerosis ofatumumab follicular lymphoma (eg,refractory or relapsed) diffuse large B cell lymphoma (eg, relapsed)chronic lymphocytic leukaemia (eg, first line therapy or relapsed) IL-23Crohns disease; tildrakizumab Inflammatory disease; Psoriasis BAFFAutoimmune disease Belimumab Neutrokine alpha systemic lupus Benlysta  ™erythematosus Tabalumab Multiple myeloma vasculitis IL5 Asthmamepolizumab IL6 Inflammatory disease sirukumab rheumatoid arthritisLp-PLA2 Atherosclerosis darapladib diabetic macular oedema CCR9chemokine receptor Inflammatory disease Vercirnon rheumatoid arthritisCrohn's disease DOPA decarboxylase Parkinson's Disease Patrome Her2Cancer, eg, gastric cancer, Tyverb  ™ EGFR breast cancer, head andTykerb  ™ neck squamous cell cancer, lapatinib ADP receptorCardiovascular disease or Brilinta condition Brilique Thrombosis, eg,arterial thrombosis VEGFR Cancer, eg, Caprelsa EGFR medullary thyroidcancer LABA Respiratory disease or PT003 GFF LAMA condition,eg, COPDFactor Xa thromboembolism apixaban OX40 ligand Asthma OxelumabGraft-versus-host disease CTLA-4 Cancer, eg, melanoma Tremelimumab CD152Autoimmune disease Ticilimumab ipilimumab PD-L1 Cancer, eg, solidtumour, MPDL3280A kidney cancer, lung cancer, MEDI4736 melanoma, NSCLC,multiple myeloma Autoimmune disease PD -1 Cancer, eg, solid tumour,Nivolumab kidney cancer, lung cancer, Keytruda melanoma, NSCLC, AMP-514multiple myeloma AMP-224 Autoimmune disease LIGHT TNFSF14 CD40 ligandJAK Inflammatory disease, eg, tofacitinib rheumatoid arthritis,psoriasis, chrohn's disease, IBD, ulcerative colitis, PsoriaticArthritis Nerve Growth Factor Pain tanezumab Osteoarthritis pain Her1receptor Cancer, eg, Non-Small Cell dacomitinib Her2 receptor LungCancer Her4 receptor c-MET Cancer, eg, Non-Small Cell crizotinib ALKLung Cancer Programmed cell death 1 receptor Cancer, eg, renal cellNivolumab carcinoma SLAMF7 Cancer, eg, multiple Elotuzumab CD319 myelomaCD30 Cancer, eg, Hodgkin Brentuximab lymphoma, systemic Brentuximabvedotin anaplastic large cell lymphoma, T-cell lymphoma GPR40 Diabetes,eg, diabetes Fasiglifam DPP-4 mellitus trelagliptin VEFGR-1 receptorCancer, eg, non-squamous Motesanib VEFGR-2 receptor non-small cell lungcancer Motesanib diphosphate VEFGR-3 receptor PDGFR cKit amyloid βAlzheimer's disease Solanezumab TNF alpha Inflammatory disease, eg,SIMPONI  ™ rheumatoid arthritis, HUMIRA  ™ psoriasis, chrohn's disease,REMICADE  ™ IBD, ulcerative colitis, ENBREL  ™ Psoriatic ArthritisAdalimumab IL-21 Autoimmune disease or Agonist or antagonist conditionantibody specific for Inflammatory disease or human IL-21 conditionNNC0114-0005 Rheumatoid arthritis NNC0114-0006 Crohn's disease NN8828IBD ATR-107 Ulcerative colitis Said or an anti-IL-21 Systemic lupusantibody in combination erythematosus (SLE) with an agent selected fromGraft versus host disease the group consisting of Cancer ipilimumab (eg,to treat Metastatic melanoma melanoma), an anti-PD1 Renal cell carcinomaantibody (eg, to treat solid Melanoma tumours), sunitinib (eg, to Solidtumours treat renal cell carcinoma), Acute myeloid leukaemia rituximab(eg, to treat Non- Non-Hodgkin's lymphoma Hodgkin's lymphoma), Ovariancancer sorafenib (eg, to treat renal Colorectal cancer cell carcinoma),doxorubicin (eg, to treat ovarian cancer) and cetuximab (eg, to treatcolorectal cancer). TIGIT immuno-oncology condition DMAM-1(immuno-oncology) CD96 (immuno-oncology) DR3 (agonist forimmuno-oncology) TL1A, DR3 (blocker for inflammatory disease) DMAM-1immuno-oncology condition CD96 immuno-oncology condition DR3immuno-oncology condition Agonist inflammatory disease Antagonist TL1Ainflammatory disease TIM3 CD200 CSF1R LAG3 GITR CD40

TABLE 6 PCSK9 SEQUENCES FORM/ ALLELE VERSION SEQUENCE AMINO ACIDSEQUENCES Italics = signal sequence 1-30.

lower case = catalytic domain 153-449 UPPER CASE = C-terminal domain450-692 Underlined = residues changed from allel a in other sequences(aa residue number shown) a Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

a Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

a Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

f Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

f Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

f, p, aj Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

c Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

c, q Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

r Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

r Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

r Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

p Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

p Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

m Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

m Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

m Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

e Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

e Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

e Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

h Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

h Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

h Maturesipwnleritppryadeyqppdggslvevylldtsiqsdhreiegrvmvtdfenvpeedgtrfhrqaskcdshgformthiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

aj Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

aj Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

q Pro-Form with

Signal

Sequence

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

q Pro-Form

thiagvvsgrdagvakgasmrslrvlncqgkgtvsgtllglefirksqivqpvgpivvllplaggysrvlnaacqrlaragvvlvtaagnfrddaclyspasapevitvgatnaqdqpvtigtigtnfgrcvdlfapgediigassdcstcfvsqsgtsqaaahvagiaammisaepeitlaelrqrllhfsak

EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLTGCSSHWEVEDLGT

NUCLEOTIDE SEQUENCES Italics = nucleotide sequence encoding signalsequence (nucleotides 1-90) Courier = nucleotide sequence encoding propeptide (nucleotides 91-456) lowercase = nucleotide sequence encodingcatalytic domain (nucleotides 457-1346) UPPER CASE = nucleotide sequenceencoding C-terminal domain (nucleotides 1347-2076) aATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC fATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC cATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC rATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC pATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC mATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC eATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC hATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC ajATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC qATGGGCACCGTCAGCTCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCTCCTGGGTC

cggtaccgggcggatgaataccagccccccgacggaggcagcctggtggaggtgtatctcctagacaccagcatacagagtgaccaccgggaaatcgagggcagggtcatggtcaccgacttcgagaatgtgcccgaggaggacgggacccgcttccacagacaggccagcaagtgtgacagtcatggcacccacctggcaggggtggtcagcggccgggatgccggcgtggccaagggtgccagcatgcgcagcctgcgcgtgctcaactgccaagggaagggcacggttagcggcaccctcataggcctggagtttattcggaaaagccagctggtccagcctgtggggccactggtggtgctgctgcccctggcgggtgggtacagccgcgtcctcaacgccgcctgccagcgcctggcgagggctggggtcgtgctggtcaccgctgccggcaacttccgggacgatgcctgcctctactccccagcctcagctcccgaggtcatcacagttggggccaccaatgcccaagaccagccggtgaccctggggactttggggaccaactttggccgctgtgtggacctctttgccccaggggaggacatcattggtgcctccagcgactgcagcacctgctttgtgtcacagagtgggacatcacaggctgctgcccacgtggctggcattgcagccatgatgctgtctgccgagccggagctcaccctggccgagttgaggcagagactg

cacccatGGGGCAGGTTGGCAGCTGTTTTGCAGGACTGTATGGTCAGCACACTCGGGGCCTACACGGATGGCC

CGGGGCGAGCGCATGGAGGCCCAAGGGGGCAAGCTGGTCTGCCGGGCCCACAACGCTTTTGGGGGTGAGGGTGTCTACGCCATTGCCAGGTGCTGCCTGCTACCCCAGGCCAACTGCAGCGTCCACACAGCTCCACCAGCTGAGGCCAGCATGGGGACCCGTGTCCACTGCCACCAACAGGGCCACGTCCTCACAGGCTGCAGCTCCCACTGGGAGGTGGAGGACCTTGGCACCCACAAGCCGCCTGTGCTGAGGCCACGAGGTCAGCCCAACCAGTGCGTGGGCCACAGGGAGGCCAGCATCCACGCTTCCTGCTGCCATGCCCCAGGTCTGGAATGCAAAGTCAAGGAGCATGGAA

CTGGGACCTCCCACGTCCTGGGGGCCTACGCCGTAGACAACACGTGTGTAGTCAGGAGCCGGGACGTCAGCA

CCTCCCAGGAGCTCCAGTGAC

TABLE 7 Human VH3-23 Variant Alleles Cumulative VH3-23 allele haplotypefrequency SNPs a (=VH3-23*04) 0.0983 rs56069819 d 0.0087 rs56069819rs61750837 rs61752504 e 0.0046 rs56069819 rs1064090 rs1055799 j 0.0009rs56069819 rs1055799 u 0.0005 rs56069819 rs1064091 s 0.0005 rs56069819rs1064091 rs61752504 rs61750837 r 0.0005 rs56069819 rs1064090 TOTAL:0.114

TABLE 8 Exemplary anti-PCSK9 antibodies and/or antibody fragments SEQ IDNOs comprising an anti-PCSK9 monoclonal antibody or fragment thereofPatent or patent Pblication Light chain complementary determiningregions US20120020975 A1 (CDRL) SEQ ID NO: 5, 7, 9, 10, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37,38, 39, 40, 42, 44, 46, 270, 271, 272, 273, 275, 277, 286, 287, 288,297, 299, 301, 405, 407, 409, 411, 413, 415, 417, 421, 425, 429, 433,437, 441, 445, 449, 453, 457, 461, 465, 469, 473, 477, 481, 485; Heavychain complementary determining regions (CDRH) SEQ ID NO: 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 62, 64, 65, 67, 69, 71, 72,74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 278, 289, 290, 291, 292,298, 300, 302, 401, 404, 406, 408, 410, 412, 414, 416, 419, 423, 427,431, 435, 439, 443, 447, 451, 455, 459, 463, 467, 471, 475, 479, 483;CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120027765A1 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, 46, 405, 407, 409, 411, 413, 415, 417, 465; CDRH SEQ ID NO: 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72,74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412,414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US8168762B217, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38,39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 465; CDRH SEQ IDNO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67,69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414, 416, 463; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15,16, US20120020976A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 222, 229, 238, 405, 407, 409,411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81,83, 85, 87, 89, 91, 247, 256, 265, 404, 406, 408, 410, 412, 414, 416;CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130085265A1 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ IDNO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67,69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406,408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12,13, 15, 16, US20130079501A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80,81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414, 416, 459, 463,483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120213797A1 17,18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39,40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 158, 162, 395, 473,477; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60,62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89,91, 404, 406, 408, 410, 412, 414, 416, 180, 175, 308, 368, 471, 475;CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20120251544A1 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, 46, 405, 407, 409, 411, 413, 415, 417; CDRH SEQ ID NO: 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74,76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412,414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, US20130052201A117, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38,39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417, 461, 465, 485;CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62,64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91,404, 406, 408, 410, 412, 414, 416, 459, 463, 483; CDRL SEQ ID NO: 5, 7,9, 10, 12, 13, 15, 16, US20130058944A1 17, 18, 19, 20, 21, 22, 23, 24,26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407,409, 411, 413, 415, 417, 461, 465, 485; CDRH SEQ ID NO: 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76,77, 78, 79, 80, 81, 83, 85, 87, 89, 91, 404, 406, 408, 410, 412, 414,416, 459, 463, 483; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,US20130079502A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413, 415, 417;CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 62,64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85, 87, 89, 91,404, 406, 408, 410, 412, 414, 416; CDRL SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, US20130245235A1 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31,32, 33, 35, 36, 37, 38, 39, 40, 42, 44, 46, 405, 407, 409, 411, 413,415, 417; CDRH SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 60, 62, 64, 65, 67, 69, 71, 72, 74, 76, 77, 78, 79, 80, 81, 83, 85,87, 89, 91, 404, 406, 408, 410, 412, 414, 416;

TABLE 9 Human variable & constant variants distributed over severalhuman ethnic populations - useful for ligand tailoring Nucleotide HornHuman Amino Acid Variation² Freq⁷ Gene Example Position (NCBl dbSNPVariant (Het + Segment Human & reference Nucleotide Human No. Het HornCum Type Allele¹ Variation number)³ Position Populations⁴ Individs⁵Freq⁶ freq⁸) Freq⁹ IGHG1 IGHG1*01 204D GAT 14:106208086 A 153 0.4000.096 0.296 (CH3 (forward strand) (European (0.496) variation) ancestry)IGHG1*03 204E GAG 14:106208086 A 366 0.400 0.504 0.704 (CH3 (rs1045853)(forward strand) (European (0.904) variation) ancestry IGHG1*01 206L CTG14:106208082 A 0.358 0.104 0.283 (CH3 (forward strand) (European (0.462)variation) ancestry) IGHG1*03 206M ATG 14:106208082 A 0.358 0.538 0.717(CH3 (rs11621259) (forward strand) (European (0.896) variation)ancestry) IGHG2 IGHG2*01 72P CCC 14:106110914 B 0.336 0.540 0.708 (CH1(forward strand) (0.876) variation) IGHG2*02 72T ACC 14:106110914 B0.336 0.124 0.292 (CH1 (rs11627594) (forward strand) variation) IGHG2*0175N AAC 14:106110904 A 0.007 0.993 0.997 (CH1 (forward strand)variation) IGHG2*04 75S AGC 14:106110904 A 0.007 0.004 (CH1(rs201590297) (forward strand) variation) IGHG2*01 76F TTC (CH1variation) IGHG2*04 76L TTG (CH1 variation) IGHG2*01 161V GTG14:10611013 B 0.342 0.539 0.711 (CH2 (forward strand) (0.881) variation)IGHG2*02 161M ATG 14:10611013 B 0.342 0.118 0.289 (CH2 (rs8009156)(forward strand) (0.46) variation) IGHG2*01 257A GCC 14:106109752 C0.199 0.493 0.592 (CH3 (forward strand) (0.692) variation) D 0.007 0.9920.995 (0.999) IGHG2*06 257S TCC 14:106109752 C 0.199 0.308 0.408 (CH3(rs4983499) (forward strand) (0.507) variation) D 0.007 0.002 0.005(0.009) IGHG4 IGHG4*01 189L CTG 14:105624992 B 1047 0.315 0.644 0.801(CH2 (forward strand) (0.959) variation) IGHG4*02 189V GTG 14:105624992B 389 0.315 0.041 0.199 (CH2 (rs8015545) (forward strand) (0.356)variation) IGHG4*01 289R AGG 14:105624594 (CH3 (forward strand)variation) IGHG4*03 289K AAG 14:105624594 (CH3 (rs77498506) (forwardstrand) variation) Table Footnotes: ¹IMGT notation (ww.imgt.org); referto figures for other alleles comprising this variation. ²SNP Underlinedin Codon. ³NCBI dbSNP Build 138 released on Apr 25, 2013. ⁴Humanpopulation used for representative variant frequency analysis.Populations  A This population included 662 participants of Europeandescent from the ClinSeq project, all of whom had undergone whole-exomesequencing using Agilent's 38Mb or 50Mb capture kit.  B 1000 Genomesdatabase.  C ESP6500:African_American.  D ESP6500:European_American.⁵Number of individuals in representative population found to have theallele. ⁶Heterozygous human genotype frequency, ie, cumulative frequencyof all genotypes having one occurrence of the variant allele and oneoccurrence of another allele (heterozygous state), eg, ac genotype inthe population. ⁷Homozygous human genotype frequency, ie, cumulativefrequency of two occurrences of the variant allele (homozygous state),eg, cc genotype in the population. ⁸Total human genotype frequency, ie,total of heterozygous plus homozygous human genotype frequencies.⁹Cumulative human allele frequency of all occurrences of the variantallele in the population.

TABLE 10 FURTHER SEQUENCES SEQ Human ID NO: AlleleNucleotide/Amino Acid Sequence HEAVY CHAIN ALLELES 41 IGHG1*01gcctccaccaagggcccatcggtcttccccctggcaccctcctccaaga (CH1 + Hinge +gcacctctggg CH2 + CH3 + CH −ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccgg S) tgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcc tacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgg gcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga aagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaac tcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatct cccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtca agttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggc tgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgaga aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatc ccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca accactacacgcagaagagcctctccctgtctccgggtaaa 42ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSR D E LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK D  = position 204 L  = position 206 43IGHG2*01 gcctccaccaagggcccatcggtcttccccctggcgccctgctccagga (CH1 +Hinge + gcacctccgag CH2 + CH3 + CH −agcacagccgccctgggctgcctggtcaaggactacttccccgaaccgg S) tgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcc tacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcg gcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaaga cagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggac cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg aggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggt acgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaaca gcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaagg agtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctcca aaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggaga tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcg ccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgc tggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggc agcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgc agaagagcctctccctgtctccgggtaaa 44 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTV P SS NF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG V EVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDI A VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK P  = position 72 N  = position 75F  = position 76 V  = position 161 A  = position 257 45 IGHV1-18*01caggttcagctggtgcagtctggagctgaggtgaagaagcctggggcct cagtgaaggtctcctgcaaggcttctggttacacctttaccagctatggtatcagctggg tgcgacaggcccctggacaagggcttgagtggatgggatggatcagcgcttacaatggta acacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacga gcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtg cgagaga 46QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCAR 47IGHV1-46*01 caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtt tcctgcaaggcatctggatacaccttcaccagctactatatgcactgggtgcgacaggcc cctggacaagggcttgagtggatgggaataatcaaccctagtggtggtagcacaagctac gcacagaagttccagggcagagtcaccatgaccagggacacgtccacgagcacagtctac atggagctgagcagcctgagatctgaggacacggccgtgtattactgtg cgagaga 48QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARLIGHT CHAIN ALLELES 49 IGKC*01cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca cagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag cagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgc ccgtcacaaagagcttcaacaggggagagtgt 50RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V YA C EVTHQGLSSPVTKSFNRGEC V  = position 84 C = position 87 51 IGLC2*01ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctg aggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggag ccgtgacagtggcttggaaagcagatagcagccccgtcaaggcgggagtggagaccacca caccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctg agcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtgg agaagacagtggcccctacagaatgttca 52 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 53 IGKV4-1*01atggtgttgcagacccaggtcttcatttctctgttgctctggatctctg gtgcctacggggacatcgtgatgacccagtctccagactccctggctgtgtctctgggcg agagggccaccatcaactgcaagtccagccagagtgttttatacagctccaacaataaga actacttagcttggtaccagcagaaaccaggacagcctcctaagctgctcatttactggg catctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatt tcactctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcagcaat attatagtact cctcc 54DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYST P 55IGKV1-13*02 atggacatgagggtccccgctcagctcctggggcttctgctgctctggctcccagcaggt gccagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagac agagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcag cagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggg gtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgtcaacagtttaatagttaccctcagtgc cagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacag agtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcagca gaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggt cccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcct gcagcctgaagattttgcaacttattactgtcaacagtttaatagttac cctca 57IGKJ2*01 tgtacacttttggccaggggaccaagctggagatcaaac 58 YTFGQGTKLEIK 59IGLJ2*01 tgtggtattcggcggagggaccaagctgaccgtcctag 60 VVFGGGTKLTVL 61An IGHG1*01 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGHeavy Chain VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV ConstantEPKSCDKTHTCPPCPAPELLGGPS RegionVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 An IGKC*01RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa LightGNSQESVIEQDSKDSTYSLSSILTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGECConstant Region 63 An IGHG2*01ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy ChainVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ConstantERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH RegionEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64 An IGLC2*01QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK LambdaAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT Light Chain VAPTECSConstant Region 65 An IGHG2*01ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG Heavy ChainVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV ConstantERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH RegionEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 66 An IGKC*01RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS Kappa LightGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV Chain TKSFNRGECConstant Region HUMAN IL4Ra SEQUENCES 67 Amino AcidMGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNGPTNC SequenceSTELRLLYQLVFLLSEAHTC I PENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQHLLLGVSVSCIVILAVCLLCYVSITKIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGR E GIVARLTESLFLDLLGEENGGFCQQDMGES C LLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTE TPLVIAGNPAYRSFSNS LSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGY Q EFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDR S SPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPK IVNFVSVGPTYMRVS I = position 75 E  = position 400 C  = position 431 S  = position 503 Q = position 576 S = position 752 68 NucleotideATGGGGTGGCTTTGCTCTGGGCTCCTGTTCCCTGTGAGCTGCCTGGTCCTGCTG SequenceCAGGTGGCAAGCTCTGGGAACATGAAGGTCTTGCAGGAGCCCACCTGCGTCTCCGACTACATGAGCATCTCTACTTGCGAGTGGAAGATGAATGGTCCCACCAATTGCAGCACCGAGCTCCGCCTGTTGTACCAGCTGGTTTTTCTGCTCTCCGAAGCCCACACGTGTATCCCTGAGAACAACGGAGGCGCGGGGTGCGTGTGCCACCTGCTCATGGATGACGTGGTCAGTGCGGATAACTATACACTGGACCTGTGGGCTGGGCAGCAGCTGCTGTGGAAGGGCTCCTTCAAGCCCAGCGAGCATGTGAAACCCAGGGCCCCAGGAAACCTGACAGTTCACACCAATGTCTCCGACACTCTGCTGCTGACCTGGAGCAACCCGTATCCCCCTGACAATTACCTGTATAATCATCTCACCTATGCAGTCAACATTTGGAGTGAAAACGACCCGGCAGATTTCAGAATCTATAACGTGACCTACCTAGAACCCTCCCTCCGCATCGCAGCCAGCACCCTGAAGTCTGGGATTTCCTACAGGGCACGGGTGAGGGCCTGGGCTCAGTGCTATAACACCACCTGGAGTGAGTGGAGCCCCAGCACCAAGTGGCACAACTCCTACAGGGAGCCCTTCGAGCAGCACCTCCTGCTGGGCGTCAGCGTTTCCTGCATTGTCATCCTGGCCGTCTGCCTGTTGTGCTATGTCAGCATCACCAAGATTAAGAAAGAATGGTGGGATCAGATTCCCAACCCAGCCCGCAGCCGCCTCGTGGCTATAATAATCCAGGATGCTCAGGGGTCACAGTGGGAGAAGCGGTCCCGAGGCCAGGAACCAGCCAAGTGCCCACACTGGAAGAATTGTCTTACCAAGCTCTTGCCCTGTTTTCTGGAGCACAACATGAAAAGGGATGAAGATCCTCACAAGGCTGCCAAAGAGATGCCTTTCCAGGGCTCTGGAAAATCAGCATGGTGCCCAGTGGAGATCAGCAAGACAGTCCTCTGGCCAGAGAGCATCAGCGTGGTGCGATGTGTGGAGTTGTTTGAGGCCCCGGTGGAGTGTGAGGAGGAGGAGGAGGTAGAGGAAGAAAAAGGGAGCTTCTGTGCATCGCCTGAGAGCAGCAGGGATGACTTCCAGGAGGGAAGGGAGGGCATTGTGGCCCGGCTAACAGAGAGCCTGTTCCTGGACCTGCTCGGAGAGGAGAATGGGGGCTTTTGCCAGCAGGACATGGGGGAGTCATGCCTTCTTCCACCTTCGGGAAGTACGAGTGCTCACATGCCCTGGGATGAGTTCCCAAGTGCAGGGCCCAAGGAGGCACCTCCCTGGGGCAAGGAGCAGCCTCTCCACCTGGAGCCAAGTCCTCCTGCCAGCCCGACCCAGAGTCCAGACAACCTGACTTGCACAGAGACGCCCCTCGTCATCGCAGGCAACCCTGCTTACCGCAGCTTCAGCAACTCCCTGAGCCAGTCACCGTGTCCCAGAGAGCTGGGTCCAGACCCACTGCTGGCCAGACACCTGGAGGAAGTAGAACCCGAGATGCCCTGTGTCCCCCAGCTCTCTGAGCCAACCACTGTGCCCCAACCTGAGCCAGAAACCTGGGAGCAGATCCTCCGCCGAAATGTCCTCCAGCATGGGGCAGCTGCAGCCCCCGTCTCGGCCCCCACCAGTGGCTATCAGGAGTTTGTACATGCGGTGGAGCAGGGTGGCACCCAGGCCAGTGCGGTGGTGGGCTTGGGTCCCCCAGGAGAGGCTGGTTACAAGGCCTTCTCAAGCCTGCTTGCCAGCAGTGCTGTGTCCCCAGAGAAATGTGGGTTTGGGGCTAGCAGTGGGGAAGAGGGGTATAAGCCTTTCCAAGACCTCATTCCTGGCTGCCCTGGGGACCCTGCCCCAGTCCCTGTCCCCTTGTTCACCTTTGGACTGGACAGGGAGCCACCTCGCAGTCCGCAGAGCTCACATCTCCCAAGCAGCTCCCCAGAGCACCTGGGTCTGGAGCCGGGGGAAAAGGTAGAGGACATGCCAAAGCCCCCACTTCCCCAGGAGCAGGCCACAGACCCCCTTGTGGACAGCCTGGGCAGTGGCATTGTCTACTCAGCCCTTACCTGCCACCTGTGCGGCCACCTGAAACAGTGTCATGGCCAGGAGGATGGTGGCCAGACCCCTGTCATGGCCAGTCCTTGCTGTGGCTGCTGCTGTGGAGACAGGTCCTCGCCCCCTACAACCCCCCTGAGGGCCCCAGACCCCTCTCCAGGTGGGGTTCCACTGGAGGCCAGTCTGTGTCCGGCCTCCCTGGCACCCTCGGGCATCTCAGAGAAGAGTAAATCCTCATCATCCTTCCATCCTGCCCCTGGCAATGCTCAGAGCTCAAGCCAGACCCCCAAAATCGTGAACTTTGTCTCCGTGGGACCCACATACATGAGGGTCTCTTAGANTI-HUMAN IL4Ra ANTIBODY SEQUENCES 69 VHEVQLVESGGG LEQPGGSLRLSCAGSGFTFR DYAMTWVRQA PGKGLEWVSSISGSGGNTYY ADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKDRLSITIRPRYYGLDVWGQGTTVTVSS 70 VLDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTP YTFGQGTKLEIK 71HEAVY CHAIN EVQLVESGGG LEQPGGSLRL SCAGSGFTFR DYAMTWVRQA PGKGLEWVSSISGSGGNTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDRLSITIRPRYY GLDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAALGCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSSLGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQFNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPREPQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G 72 LIGHT CHAINDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSIGYNYLDWYLQKSGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQALQTPYTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFHUMAN IGHG4 SEQUENCES 73 IGHG4*01ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG AMINO ACIDVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV SEQUENCEESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV L HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS R LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK L  = position 189 R  = position 289 74IGHG4*01 GCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGA NUCLEOTIDEGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTT SEQUENCECCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC C TGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA G GCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAA ATGA C  =position 565 ( C TG encodes 189V) G  = position 866 (A G G encodes 289R)Nav1.7 SEQUENCES 75 HUMAN Nav1.7MAMLPPPGPQSFVHFTKQSLALIEQRIAERKSKEPKEEKKDDDEEAPKPSSDLEAG AMINO ACIDKQLPFIYGDIPPGMVSEPLEDLDPYYADKKTFIVLNKGKTIFRFNATPALYMLSPFS SEQUENCEPLRRISIKILVHSLFSMLIMCTILTNCIFMTMNNPPDWTKNVEYTFTGIYTFESLVKI(ENST00000303354)LARGFCVGEFTFLRDPWNWLDFVVIVFAYLTEFVNLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVFCLSVFALIGLQLFMGNLKHKCFRNSLENNETLESIMNTLESEEDFRKYFYYLEGSKDALLCGFSTDSGQCPEGYTCVKIGRNPDYGYTSFDTFSWAFLALFRLMTQDYWENLYQQTLRAAGKTYMIFFVVVIFLGSFYLINLILAVVAMAYEEQNQANIEEAKQKELEFQQMLDRLKKEQEEAEAIAAAAAEYTSIRRSRIMGLSESSSETSKLSSKSAKERRNRRKKKNQKKLSSGEEKGDAEKLSKSESEDSIRRKSFHLGVEGHRRAHEKRLSTPNQSPLSIRGSLFSARRSSRTSLFSFKGRGRDIGSETEFADDEHSIFGDNESRRGSLFVPHRPQERRSSNISQASRSPPMLPVNGKMHSAVDCNGVVSLVDGRSALMLPNGQLLPEVIIDKATSDDSGTTNQIHKKRRCSSYLLSEDMLNDPNLRQRAMSRASILTNTVEELEESRQKCPPWWYRFAHKFLIWNCSPYWIKFKKCIYFIVMDPFVDLAITICIVLNTLFMAMEHHPMTEEFKNVLAIGNLVFTGIFAAEMVLKLIAMDPYEYFQVGWNIFDSLIVTLSLVELFLADVEGLSVLRSFRLLRVFKLAKSWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKECVCKINDDCTLPRWHMNDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLIVYMMVMVIGNLVVLNLFLALLLSSFSSDNLTAIEEDPDANNLQIAVTRIKKGINYVKQTLREFILKAFSKKPKISREIRQAEDLNTKKENYISNHTLAEMSKGHNFLKEKDKISGFGSSVDKHLMEDSDGQSFIHNPSLTVTVPIAPGESDLENMNAEELSSDSDSEYSKVRLNRSSSSECSTVDNPLPGEGEEAEAEPMNSDEPEACFTDGCVWRFSCCQVNIESGKGKIWWNIRKTCYKIVEHSWFESFIVLMILLSSGALAFEDIYIERKKTIKIILEYADKIFTYIFILEMLLKWIAYGYKTYFTNAWCWLDFLIVDVSLVTLVANTLGYSDLGPIKSLRTLRALRPLRALSRFEGMRVVVNALIGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFYECINTTDGSRFPASQVPNRSECFALMNVSQNVRWKNLKVNFDNVGLGYLSLLQVATFKGWTIIMYAAVDSVNVDKQPKYEYSLYMYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKIQGCIFDLVTNQAFDISIMVLICLNMVTMMVEKEGQSQHMTEVLYWINVVFIILFTGECVLKLISLRHYYFTVGWNIFDFVVVIISIVGMFLADLIETYFVSPTLFRVIRLARIGRILRLVKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSNFAYVKKEDGINDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPKKVHPGSSVEGDCGNPSVGIFYFVSYIIISFLVVVNMYIAVILENFSVATEESTEPLSEDDFEMFYEVWEKFDPDATQFIEFSKLSDFAAALDPPLLIAKPNKVQLIAMDLPMVSGDRIHCLDILFAFTKRVLGESGEMDSLRSQMEERFMSANPSKVSYEPITTTLKRKQEDVSATVIQRAYRRYRLRQNVKNISSIYIKDGDRDDDLLNKKDMAFDNVNENSSPEKTDATSSTTSPPSYDSVTKPDKEKYEQDR TEKEDKGKDSKESKK76 HUMAN Nav1.7 ATGGCAATGTTGCCTCCCCCAGGACCTCAGAGCTTTGTCCATTTCACAAAACANUCLEOTIDE GTCTCTTGCCCTCATTGAACAACGCATTGCTGAAAGAAAATCAAAGGAACCCASEQUENCE AAGAAGAAAAGAAAGATGATGATGAAGAAGCCCCAAAGCCAAGCAGTGACT(ENST00000303354) TGGAAGCTGGCAAACAGCTGCCCTTCATCTATGGGGACATTCCTCCCGGCATGGTGTCAGAGCCCCTGGAGGACTTGGACCCCTACTATGCAGACAAAAAGACTTTCATAGTATTGAACAAAGGGAAAACAATCTTCCGTTTCAATGCCACACCTGCTTTATATATGCTTTCTCCTTTCAGTCCTCTAAGAAGAATATCTATTAAGATTTTAGTACACTCCTTATTCAGCATGCTCATCATGTGCACTATTCTGACAAACTGCATATTTATGACCATGAATAACCCACCGGACTGGACCAAAAATGTCGAGTACACTTTTACTGGAATATATACTTTTGAATCACTTGTAAAAATCCTTGCAAGAGGCTTCTGTGTAGGAGAATTCACTTTTCTTCGTGACCCGTGGAACTGGCTGGATTTTGTCGTCATTGTTTTTGCGTATTTAACAGAATTTGTAAACCTAGGCAATGTTTCAGCTCTTCGAACTTTCAGAGTATTGAGAGCTTTGAAAACTATTTCTGTAATCCCAGGCCTGAAGACAATTGTAGGGGCTTTGATCCAGTCAGTGAAGAAGCTTTCTGATGTCATGATCCTGACTGTGTTCTGTCTGAGTGTGTTTGCACTAATTGGACTACAGCTGTTCATGGGAAACCTGAAGCATAAATGTTTTCGAAATTCACTTGAAAATAATGAAACATTAGAAAGCATAATGAATACCCTAGAGAGTGAAGAAGACTTTAGAAAATATTTTTATTACTTGGAAGGATCCAAAGATGCTCTCCTTTGTGGTTTCAGCACAGATTCAGGTCAGTGTCCAGAGGGGTACACCTGTGTGAAAATTGGCAGAAACCCTGATTATGGCTACACGAGCTTTGACACTTTCAGCTGGGCCTTCTTAGCCTTGTTTAGGCTAATGACCCAAGATTACTGGGAAAACCTTTACCAACAGACGCTGCGTGCTGCTGGCAAAACCTACATGATCTTCTTTGTCGTAGTGATTTTCCTGGGCTCCTTTTATCTAATAAACTTGATCCTGGCTGTGGTTGCCATGGCATATGAAGAACAGAACCAGGCAAACATTGAAGAAGCTAAACAGAAAGAATTAGAATTTCAACAGATGTTAGACCGTCTTAAAAAAGAGCAAGAAGAAGCTGAGGCAATTGCAGCGGCAGCGGCTGAATATACAAGTATTAGGAGAAGCAGAATTATGGGCCTCTCAGAGAGTTCTTCTGAAACATCCAAACTGAGCTCTAAAAGTGCTAAAGAAAGAAGAACAGAAGAAAGAAAAAGAATCAAAAGAAGCTCTCCAGTGGAGAGGAAAAGGGAGATGCTGAGAAATTGTCGAAATCAGAATCAGAGGACAGCATCAGAAGAAAAAGTTTCCACCTTGGTGTCGAAGGGCATAGGCGAGCACATGAAAAGAGGTTGTCTACCCCCAATCAGTCACCACTCAGCATTCGTGGCTCCTTGTTTTCTGCAAGGCGAAGCAGCAGAACAAGTCTTTTTAGTTTCAAAGGCAGAGGAAGAGATATAGGATCTGAGACTGAATTTGCCGATGATGAGCACAGCATTTTTGGAGACAATGAGAGCAGAAGGGGCTCACTGTTTGTGCCCCACAGACCCCAGGAGCGACGCAGCAGTAACATCAGCCAAGCCAGTAGGTCCCCACCAATGCTGCCGGTGAACGGGAAAATGCACAGTGCTGTGGACTGCAACGGTGTGGTCTCCCTGGTTGATGGACGCTCAGCCCTCATGCTCCCCAATGGACAGCTTCTGCCAGAGGTGATAATAGATAAGGCAACTTCTGATGACAGCGGCACGACCAATCAAATACACAAGAAAAGGCGTTGTAGTTCCTATCTCCTTTCAGAGGATATGCTGAATGATCCCAACCTCAGACAGAGAGCAATGAGTAGAGCAAGCATATTAACAAACACTGTGGAAGAACTTGAAGAGTCCAGACAAAAATGTCCACCTTGGTGGTACAGATTTGCACACAAATTCTTGATCTGGAATTGCTCTCCATATTGGATAAAATTCAAAAAGTGTATCTATTTTATTGTAATGGATCCTTTTGTAGATCTTGCAATTACCATTTGCATAGTTTTAAACACATTATTTATGGCTATGGAACACCACCCAATGACTGAGGAATTCAAAAATGTACTGCTATAGGAAATTTGGTCTTTACTGGAATCTTTGCAGCTGAAATGGTATTAAAACTGATTGCCATGGATCCATATGAGTATTTCCAAGTAGGCTGGAATATTTTTGACAGCCTTATTGTGACTTTAAGTTTAGTGGAGCTCTTTCTAGCAGATGTGGAAGGATTGTCAGTTCTGCGATCATTCAGACTGCTCCGAGTCTTCAAGTTGGCAAAATCCTGGCCAACATTGAACATGCTGATTAAGATCATTGGTAACTCAGTAGGGGCTCTAGGTAACCTCACCTTAGTGTTGGCCATCATCGTCTTCATTTTTGCTGTGGTCGGCATGCAGCTCTTTGGTAAGAGCTACAAAGAATGTGTCTGCAAGATCAATGATGACTGTACGCTCCCACGGTGGCACATGAACGACTTCTTCCACTCCTTCCTGATTGTGTTCCGCGTGCTGTGTGGAGAGTGGATAGAGACCATGTGGGACTGTATGGAGGTCGCTGGTCAAGCTATGTGCCTTATTGTTTACATGATGGTCATGGTCATTGGAAACCTGGTGGTCCTAAACCTATTTCTGGCCTTATTATTGAGCTCATTTAGTTCAGACAATCTTACAGCAATTGAAGAAGACCCTGATGCAAACAACCTCCAGATTGCAGTGACTAGAATTAAAAAGGGAATAAATTATGTGAAACAAACCTTACGTGAATTTATTCTAAAAGCATTTTCCAAAAAGCCAAAGATTTCCAGGGAGATAAGACAAGCAGAAGATCTGAATACTAAGAAGGAAAACTATATTTCTAACCATACACTTGCTGAAATGAGCAAAGGTCACAATTTCCTCAAGGAAAAAGATAAAATCAGTGGTTTTGGAAGCAGCGTGGACAAACACTTGATGGAAGACAGTGATGGTCAATCATTTATTCACAATCCCAGCCTCACAGTGACAGTGCCAATTGCACCTGGGGAATCCGATTTGGAAAATATGAATGCTGAGGAACTTAGCAGTGATTCGGATAGTGAATACAGCAAAGTGAGATTAAACCGGTCAAGCTCCTCAGAGTGCAGCACAGTTGATAACCCTTTGCCTGGAGAAGGAGAAGAAGCAGAGGCTGAACCTATGAATTCCGATGAGCCAGAGGCCTGTTTCACAGATGGTTGTGTATGGAGGTTCTCATGCTGCCAAGTTAACATAGAGTCAGGGAAAGGAAAAATCTGGTGGAACATCAGGAAAACCTGCTACAAGATTGTTGAACACAGTTGGTTTGAAAGCTTCATTGTCCTCATGATCCTGCTCAGCAGTGGTGCCCTGGCTTTTGAAGATATTTATATTGAAAGGAAAAAGACCATTAAGATTATCCTGGAGTATGCAGACAAGATCTTCACTTACATCTTCATTCTGGAAATGCTTCTAAAATGGATAGCATATGGTTATAAAACATATTTCACCAATGCCTGGTGTTGGCTGGATTTCCTAATTGTTGATGTTTCTTTGGTTACTTTAGTGGCAAACACTCTTGGCTACTCAGATCTTGGCCCCATTAAATCCCTTCGGACACTGAGAGCTTTAAGACCTCTAAGAGCCTTATCTAGATTTGAAGGAATGAGGGTCGTTGTGAATGCACTCATAGGAGCAATTCCTTCCATCATGAATGTGCTACTTGTGTGTCTTATATTCTGGCTGATATTCAGCATCATGGGAGTAAATTTGTTTGCTGGCAAGTTCTATGAGTGTATTAACACCACAGATGGGTCACGGTTTCCTGCAAGTCAAGTTCCAAATCGTTCCGAATGTTTTGCCCTTATGAATGTTAGTCAAAATGTGCGATGGAAAAACCTGAAAGTGAACTTTGATAATGTCGGACTTGGTTACCTATCTCTGCTTCAAGTTGCAACTTTTAAGGGATGGACGATTATTATGTATGCAGCAGTGGATTCTGTTAATGTAGACAAGCAGCCCAAATATGAATATAGCCTCTACATGTATATTTATTTTGTCGTCTTTATCATCTTTGGGTCATTCTTCACTTTGAACTTGTTCATTGGTGTCATCATAGATAATTTCAACCAACAGAAAAAGAAGCTTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAGCTGGGGTCCAAGAAGCCACAAAAGCCAATTCCTCGACCAGGGAACAAAATCCAAGGATGTATATTTGACCTAGTGACAAATCAAGCCTTTGATATTAGTATCATGGTTCTTATCTGTCTCAACATGGTAACCATGATGGTAGAAAAGGAGGGTCAAAGTCAACATATGACTGAAGTTTTATATTGGATAAATGTGGTTTTTATAATCCTTTTCACTGGAGAATGTGTGCTAAAACTGATCTCCCTCAGACACTACTACTTCACTGTAGGATGGAATATTTTTGATTTTGTGGTTGTGATTATCTCCATTGTAGGTATGTTTCTAGCTGATTTGATTGAAACGTATTTTGTGTCCCCTACCCTGTTCCGAGTGATCCGTCTTGCCAGGATTGGCCGAATCCTACGTCTAGTCAAAGGAGCAAAGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGTTGTTTAACATCGGCCTCCTGCTCTTCCTGGTCATGTTCATCTACGCCATCTTTGGAATGTCCAACTTTGCCTATGTTAAAAAGGAAGATGGAATTAATGACATGTTCAATTTTGAGACCTTTGGCAACAGTATGATTTGCCTGTTCCAAATTACAACCTCTGCTGGCTGGGATGGATTGCTAGCACCTATTCTTAACAGTAAGCCACCCGACTGTGACCCAAAAAAAGTTCATCCTGGAAGTTCAGTTGAAGGAGACTGTGGTAACCCATCTGTTGGAATATTCTACTTTGTTAGTTATATCATCATATCCTTCCTGGTTGTGGTGAACATGTACATTGCAGTCATACTGGAGAATTTTAGTGTTGCCACTGAAGAAAGTACTGAACCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGGGAGAAGTTTGATCCCGATGCGACCCAGTTTATAGAGTTCTCTAAACTCTCTGATTTTGCAGCTGCCCTGGATCCTCCTCTTCTCATAGCAAAACCCAACAAAGTCCAGCTCATTGCCATGGATCTGCCCATGGTTAGTGGTGACCGGATCCATTGTCTTGACATCTTATTTGCTTTTACAAAGCGTGTTTTGGGTGAGAGTGGGGAGATGGATTCTCTTCGTTCACAGATGGAAGAAAGGTTCATGTCTGCAAATCCTTCCAAAGTGTCCTATGAACCCATCACAACCACACTAAAACGGAAACAAGAGGATGTGTCTGCTACTGTCATTCAGCGTGCTTATAGACGTTACCGCTTAAGGCAAAATGTCAAAAATATATCAAGTATATACATAAAAGATGGAGACAGAGATGATGATTTACTCAATAAAAAAGATATGGCTTTTGATAATGTTAATGAGAACTCAAGTCCAGAAAAAACAGATGCCACTTCATCCACCACCTCTCCACCTTCATATGATAGTGTAACAAAGCCAGACAAAGAGAAATATGAACAAGACAGAACAGAAAAGGAAGACAAAGGGAAAGACAGCAAGGAAAGCAAAAAATAG

TABLE 11 Human IL4Ra variants distributed over several human ethnicpopulations & having desired total human genotype frequency (a) Aminoacid variability, population distributions and frequencies: Hom Freq⁴No. No Unique (Het + Hom Human Populations Individs¹ Pops² Het Freq³freq⁵) Cum Freq⁶ Most 75I 835 0.462 0.302 0.533 common (0.764) Variant75V ASW, CEU, CHB, CHS 762 14 0.462 0.235 0.467 CLM, FIN, GBR, IBS,(0.697) JPT, LWK, MXL, PUR, TSI, YRI Most 400E 988 0.256 0.648 0.777common (0.904) Variant 400A ASW, CEU, CHB, CHS 384 14 0.256 0.095 0.223CLM, FIN, GBR, IBS, (0.351) JPT, LWK, MXL, PUR, TSI, YRI Most 431C 10770.176 0.810 0.898 common (0.986) Variant 431R ASW, CEU, CHB, CHS, 207 140.176 0.014 0.102 CLM, FIN, GBR, IBS, (0.19) 431 JPT, LWK, MXL, PUR,TSI, YRI Most 503S 1010 0.269 0.656 0.79 common (0.925) Variant 503PASW, CEU, CHB, CHS, 376 14 0.269 0.075 0.21 CLM, FIN, GBR, IBS, (0.344)JPT, LWK, MXL, PUR, TSI, YRI Most 576Q 885 0.311 0.499 0.655 common(0.81) Variant 576R ASW, CEU, CHB, CHS, 547 14 0.311 0.190 0.345 CLM,FIN, GBR, IBS, (0.501) JPT, LWK, MXL, PUR, TSI, YRI Most 752S 1059 0.1760.794 0.882 common (0.97) Variant 752A ASW, CEU, CHB, CHS, 225 14 0 1760.030 0.118 CLM , FIN, GBR, IBS, (0.206) JPT, LWK, MXL, PUR, TSI, YRITable Footnotes: ¹Number of individuals in 1000 Genomes database(20110521) found to have the allele; ²Number of unique human ethnicpopulations in 1000 Genomes database in which the allele was found tooccur; ³Heterozygous human genotype frequency, ie, cumulative frequencyof all genotypes having one occurrence of the variant allele and oneoccurrence of another allele (heterozygous state), eg, ac genotype in1000 Genomes database; ⁴Homozygous human genotype frequency, ie,cumulative frequency of two occurrences of the variant allele(homozygous state), eg, cc genotype in 1000 Genomes database; and ⁵Totalhuman genotype frequency, ie, total of heterozygous plus homozygoushuman genotype frequencies. ⁶Cumulative human allele frequency of alloccurrences of the variant allele in 1000 Genomes database. (b)Nucleotide Sequence Variations of Selected Alleles Common A A T T A TAllele NucleotidePosition¹ VariantAllele 16:27344882 16:2736255116:27362643 16:27362859 16:27363079 16:27363606 Non-SynonymousNucleotide Variation² G C C C G G VariantID³ rs1805010 rs1805011rs1805012 rs1805015 rs1801275 rs1805016 CorrespondingAmino AcidVariation 75V 400A 431R 503P 576R 752A Table Footnotes: ¹Notation ischromosome number (all positions are on human chromosome 1):coordinatenumber (Ensembl release 73 - September 2013, Genome assembly: GRCh37(GCA_000001405.13), (forward strand); ²Nucleotide change (compared tomost common allele) giving rise to an amino acid change in the variantform (compared to most common allele); and ³NCBI dbSNP reference number(NCBI dbSNP Build 138 released on Apr 25, 2013).

TABLE 12 Human Nav1.7 (SCN9A) variants distributed over several humanethnic populations & having desired total human genotype frequency (a)Amino acid variability, population distributions and frequencies: HomFreq⁴ Human No. No Unique (Het+Hom Populations Individs¹ Pops² Het Freq³freq⁵) Cum Freq⁶ VARIANTS ASSOCIATED WITH PRIMARY ERYTHERMALGIA (PE)7Most 136V common Variant 136I Most 216F common Variant 216S Most 241Scommon Variant 241T Most 395N common Variant 395K Most 848I commonVariant 848T Most 858L common Variant 858H 858F Most 863A common Variant863P Most 1449F common Variant 1449V VARIANTS ASSOCIATED WITH PAROXYSMALEXTREME PAIN DISORDER(PEPD)⁷ Most 996R common Variant 996C Most 1298Vcommon Variant 1298F 1298D Most 1299V common Variant 1299F Most 1461Icommon Variant 1461T Most 1462F common Variant 1462V Most 1464T commonVariant 1464I Most 1627M common Variant 1627K VARIANTS ASSOCIATED WITHCHANNELOPATHY-ASSOCIATED INSENSITIVITY TO PAIN (CIP)⁷ Most 277R commonVariant 277X⁸ Most 328Y common Variant 328X Most 395N common Variant395K Most 459S common Variant 459X Most 693E common Variant 693X Most767I common Variant 767X Most 830R common Variant 830X Most 897W commonVariant 897X Most 1200F common Variant 1200L Most 1235I common Variant1235L Most 1488R common Variant 1488X Most 1659K common Variant 1659XMost 1689W common Variant 1689X FURTHER VARIANTS Most 422E 962 0.4420.439 0.660 common (0.881) Variant 422D ASW, CEU, CHB, CHS, 613 14 0.4420.119 0.340 CLM, FIN, GBR, IBS, (0.561) J PT, LWK, MXL, PUR, TSI, YRIMost 490S 1086 0.076 0.918 0.957 common (0.994) Variant 490N ASW, CEU,CHB, CHS, 89 14 0.076 0.005 0.043 CLM, FIN, GBR, IBS, (0.081) JPT, LWK,MXL, PUR, TSI, YR Most 943M common Variant 943L ASW, CEU, CHB, CHS 14CLM, FIN, GBR, IBS, JPT, LWK, MXL, PUR, TSI, YR Most 1161R 1076 0.1890.797 0.891 common (0.986) Variant 1161W ASW, CEU, CHB, CHS, 222 140.189 0.015 0.109 CLM , FIN, GBR, IBS, (0.204) J PT, LWK, MXL, PUR, TSI,YRI Most 1919D 1076 0.109 0.876 0.931 common (0.985) Variant 1919G ASW,CEU, CHB, CHS, 135 14 0.109 0.015 0.069 CLM, FIN, GBR, IBS, (0.124) JPT,LWK, MXL, PUR, TSI, YRI Table Footnotes: ¹Number of individuals in 1000Genomes database (20110521) found to have the allele; ²Number of uniquehuman ethnic populations in 1000 Genomes database in which the allelewas found to occur; ³Heterozygous human genotype frequency, ie,cumulative frequency of all genotypes having one occurrence of thevariant allele and one occurrence of another allele (heterozygousstate), eg, ac genotype in 1000 Genomes database; ⁴Homozygous humangenotype frequency, ie, cumulative frequency of two occurrences of thevariant allele (homozygous state), eg, cc genotype in 1000 Genomesdatabase; and ⁵Total human genotype frequency, ie, total of heterozygousplus homozygous human genotype frequencies. ⁶Cumulative human allelefrequency of all occurrences of the variant allele in 1000 Genomesdatabase. ⁷See J Clin Invest. 2007 Dec;117(12):3603-9, “Mutations insodium-channel gene SCN9A cause a spectrum of human genetic paindisorders”, Drenth JP & Waxman SG (incorporated herein by reference);amino acid position numbering is as per this reference. ⁸X here andelsewhere indicate change to an unknown amino acid (ie, a change fromthe most common amino acid at that position) Amino acid positions are asper Ensembl, unless otherwise indicated. (b) Nucleotide SequenceVariations of Selected Alleles Common G T C C T T AlleleNucleotidePosition¹ Variant Allele 2:166242648 2:166198883 2:1662864692:166272746 2:166288485 2:166277030 Non-Synonymous Nucleotide variation²A C T A C G Variant ID³ rs6746030 rs3750904 rs58022607 rs4369876rs13402180 rs12478318 Corresponding Amino Acid Variation 1161W 1919G490N 1002L 422D 943L Table Footnotes: ¹Notation is chromosome number(all positions are on human chromosome 1): coordinate number (Ensemblrelease 73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13),(forward strand); ²Nucleotide change (compared to most common allele)giving rise to an amino acid change in the variant form (compared tomost common allele); and ³NCBI dbSNP reference number (NCBI dbSNP Build138 released on Apr 25, 2013).

Example 4 Rarer IL6R Variants

The present invention provides anti-IL6R ligands; and IL6R-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of IL6R, in particular human IL6R or its ligands and inscreening assays to identify other antagonists of IL6R activity. Some ofthe ligands of the invention are useful for inhibiting binding of IL6Rto IL6 and/or gp130, or inhibiting IL6R-mediated activities.

Anti-IL6R ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human IL6R, which is a commonly-occurring form (see, eg, SEQ ID NO: 78).While such therapies are useful for human patients harbouring this formof human IL6R, the inventor considered it useful to investigate thepossibility of targeting rarer—but still naturally-occurring—forms ofIL6R amongst human populations. In this way, the inventor arrived atinsight into the natural occurrences and distributions of rarer humanIL6R forms that can serve as useful targets (at the protein or nucleicacid level) for human treatment, prophylaxis and diagnosis pertinent todiseases and conditions mediated or associated with IL6R activity. Thisparticularly provides for tailored therapies, prophylaxis and diagnosisin humans that are devoid of the common IL6R gene or protein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer IL6R polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous changesat the nucleotide level that translate into one or more correspondingchanges in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor realised that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-IL6R ligand for administration to human patients fortherapy and/or prophylaxis of IL6R-mediated or associated diseases orconditions. In this way, the patient receives drugs and ligands that aretailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example the presentinventor decided to determine a set of human IL6R variants on the basisof the following criteria, these being criteria that the inventorrealised would provide for useful medical drugs and diagnostics totailored need in the human population. The inventor selected variantshaving at least 3 of the 4 following criteria:—

-   -   Naturally-occurring human IL6R variation having a cumulative        human allele frequency of 35% or less;    -   Naturally-occurring human IL6R variation having a total human        genotype frequency of about 50% or less;    -   Naturally-occurring human IL6R variation found in many different        human ethnic populations (using the standard categorisation of        the 1000 Genomes Project; see Table 14 below); and    -   Naturally-occurring human IL6R variation found in many        individuals distributed across such many different ethnic        populations.

On the basis of these criteria, the inventor identified the variantslisted in Table 13 below. The inventor's selection included, as aconsideration, selection for nucleotide variation that produced aminoacid variation in corresponding IL6R forms (ie, non-synonymousvariations), as opposed to silent variations that do not alter aminoacid residues in the target protein.

TABLE 13 Human IL6R variants distributed over several human ethnicpopulations & having desired total human genotype frequency (a) Aminoacid variability, population distributions and frequencies: Exon 9 HomFreq⁴ No. No Unique (Het + Hom Human Populations Individs¹ Pops² HetFreq³ freq⁵) Cum Freq⁶ Most 358D 0.383 0.489 0.680 common (0.872)Variant 358A YRI, ASW, GBR, TSI, 557 14 0.383 0.128 0.320 CLM, CHB, LWK,CHS (0.511) MXL, PUR, JPT, IBS, FIN, CEU (See note 7) Most 385V 0.0860.904 0.947 common (0.99)  Variant 385I LWK, ASW, YRI, PUR 105  4 0.0860.010 0.053 (see note 8) (0.096) Table Footnotes: ¹Number of individualsin 1000 Genomes database (20110521) found to have the allele; ²Number ofunique human ethnic populations in 1000 Genomes database in which theallele was found to occur; ³Heterozygous human genotype frequency, ie,cumulative frequency of all genotypes having one occurrence of thevariant allele and one occurrence of another allele (heterozygousstate), eg, ac genotype in 1000 Genomes database; ⁴Homozygous humangenotype frequency, ie, cumulative frequency of two occurrences of thevariant allele (homozygous state), eg, cc genotype in 1000 Genomesdatabase; and ⁵Total human genotype frequency, ie, total of heterozygousplus homozygous human genotype frequencies. ⁶Cumulative human allelefrequency of all occurrences of the variant allele in 1000 Genomesdatabase. ⁷According to the 1000 Genomes database version 20130502, thisvariant is found in the following 26 human populations (KHV, GBR, CHB,CDX, CLM, MXL, CHS, JPT, GIH, PUR, ESN, FIN, ACB, BEB, YRI, ASW, ITU,PJL, TSI, PEL, MSL, LWK, STU, GWD, IBS, CEU) and in 1218 individualswith a cumulative frequency of 0.29. ⁸According to the 1000 Genomesdatabase version 20130502, this variant is found in the following 10human populations (ASW, YRI, PEL, MSL, LWK, GWD, PUR, IBS, ESN, ACB) andin 267 individuals with a cumulative frequency of 0.06. (b) NucleotideSequence Variations of Selected Alleles Common Allele A G Variant AlleleNucleotide Position¹ 1:154426970 1:154427050 Non Synonymous NucleotideVariation² C A Variant ID³ rs2228145 rs28730736 Corresponding Amino AcidVariation 358A 385I Table Footnotes: ¹Notation is chromosome number (allpositions are on human chromosome 1): coordinate number (Ensembl release73 - September 2013, Genome assembly: GRCh37 (GCA_000001405.13);²Nucleotide change (compared to most common allele) giving rise to anamino acid change in the variant form (compared to most common allele);and ³NCBI dbSNP reference number (NCBI dbSNP Build 138 released on Apr.25, 2013).

Tailoring Antibodies to Rarer IL6R Variant Profile

As outlined above, the invention includes the possibility to tailortreatment of humans further by selecting antibody-based ligands withvariable and/or constant domains based on gene segments found in manyhumans of the ethnic populations where the variant IL6R forms are foundto meet the selection criteria of the invention. An example is providedfor ligands comprising antibody VH domains derived from recombination ofhuman IGHV gene segments comprising selected nucleotides at positions inthe HCDR1 or FW3 where there is variability in humans (ie, where SNPsoccur in humans).

The inventor analysed human IGHV variation and used this to chooseligands based on human IGHV alleles comprising said selected nucleotidesand for matching to human recipient genotypes and/or phenotypes. Theinventor identified utility in using gene VH gene segments encoding (i)a CDR1 comprising a Phe at position 4 shown in SEQ ID NO: 109 andwherein said human comprises a VH gene segment encoding a CDR1comprising a Phe at position 4 shown in SEQ ID NO: 109, or the humanexpresses VH domains that comprise a CDR1 comprising a Phe at position 4shown in SEQ ID NO: 109; or (ii) a FW3 comprising a Thr at position 33shown in SEQ ID NO: 111 and wherein said human comprises a VH genesegment encoding a FW3 comprising a Thr at position 33 shown in SEQ IDNO: 111 or the human expresses VH domains that comprise a FW3 comprisinga Thr at position 33 shown in SEQ ID NO: 111. Further information isprovided in Table 14, which shows variation at these positions, as wellas the variant distributions across the 1000 Genomes Project databaserelating to many human populations.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ or constant region gene segments (see further Table 16 forrepresentative variants).

In an example, following this guidance, the chosen ligand can besarilumab.

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(ii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 113 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 113, or thehuman expresses Vκ domains that comprise a CDR3 comprising a Pro atposition 7 shown in SEQ ID NO: 113; or (ii) a FW3 comprising a Ser atposition 15 shown in SEQ ID NO: 115 and wherein said human comprises aVκ gene segment encoding a FW3 comprising a Ser at position 15 shown inSEQ ID NO: 115 or the human expresses Vκ domains that comprise a FW3comprising a Ser at position 15 shown in SEQ ID NO: 115.(iv) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 81 or aLeu at position 206 shown in SEQ ID NO: 81 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 81 or a Leu at position 206 shown in SEQ ID NO: 81.(v) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 83, an Asn at position 75shown in SEQ ID NO: 83, a Phe at position 76 shown in SEQ ID NO: 83, aVal at position 161 shown in SEQ ID NO: 83 and an Ala at position 257shown in SEQ ID NO: 83 and wherein said human comprises (i) an IGHG2*01human heavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 83, Asnat position 75 shown in SEQ ID NO: 83, Phe at position 76 shown in SEQID NO: 83, Val at position 161 shown in SEQ ID NO: 83 or Ala at position257 shown in SEQ ID NO: 83.(vi) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 93 or a Cys atposition 87 shown in SEQ ID NO: 93 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 93 ora Cys at position 87 shown in SEQ ID NO: 93.(vii) wherein the ligand comprises a human IGLC1*01 lambda chainconstant region and wherein said human comprises (i) a human IGLC1*01lambda chain constant region gene segment, or the human expressesantibodies comprising human IGLC1*01 lambda chain constant regions.

For example, as per example (iv), the inventor identified thepossibility of addressing the rarer IGH-gamma-1 SNPs 204D (observedcumulative frequency of 0.296) and 206L (observed cumulative frequencyof 0.283) individually or in combination. These residues are part of theCH3 domain, and as such they form part of antibody Fc regions. Thus,matching of these CH3 variations with the patient is especiallybeneficial for reasons as discussed above. Thus, in this example theligand of the invention comprises or consists of an antibody thatcomprises a human gamma-1 heavy chain constant region that comprises anAsp corresponding to position 204 of SEQ ID NO: 81 or a Leucorresponding to position 206 of SEQ ID NO: 81 and wherein the genome ofthe human comprises a gamma-1 heavy chain constant region nucleotidesequence that encodes such an Asp or Leu or the human expressesantibodies comprising human gamma-1 constant regions comprising such anAsp or Leu. An example of such a ligand is sarilumab.

In another example, as per example (v), the inventor identified thepossibility of addressing IGH-gamma-2 SNPs. This included considerationof Fc region variation—in this respect, the inventor focused onpositions 161 and 257 which are in the Fc region. Thus, in this examplethe ligand of the invention comprises or consists of an antibody thatcomprises a human gamma-2 heavy chain constant region that comprises anamino acid selected from the group consisting of a Pro corresponding toposition 72 of SEQ ID NO: 83, an Asn corresponding to position 75 of SEQID NO: 83, a Phe corresponding to position 76 of SEQ ID NO: 83, a Valcorresponding to position 161 of SEQ ID NO: 83 and an Ala correspondingto position 257 of SEQ ID NO: 83; and wherein the genome of the humancomprises a gamma-2 heavy chain constant region nucleotide sequence thatencodes such a selected amino acid or the human expresses antibodiescomprising human gamma-2 constant regions comprising such a selectedamino acid.

In another example, as per example (vi), the inventor addressed humankappa constant region variation. Thus, in this example the ligand of theinvention comprises or consists of an antibody that comprises a humankappa light chain constant region that comprises a Val corresponding toposition 84 of SEQ ID NO: 93 or a Cys corresponding to position 87 ofSEQ ID NO: 93; and wherein the genome of the human comprises a kappalight chain constant region nucleotide sequence that encodes such a Valor Cys or the human expresses antibodies comprising human kappa lightchain constant regions comprising such a Val or Cys. An example of sucha ligand is sarilumab.

In another example, as per example (vii), the inventor addressed humanlambda constant region variation. Thus, in this example the ligand ofthe invention comprises or consists of an antibody that comprises ahuman IGLC2*01 light chain constant region; and wherein the genome ofthe human comprises a human IGLC2*01 nucleotide sequence or the humanexpresses antibodies comprising human light chain IGLC2*01 constantregions.

Determination of Specific Binding of Ligands of the Invention to IL6RVariants

The specific binding of ligands of the invention to IL6R variants can beperformed using the following method.

Method of SPR Determination of Binding

Binding of the antibodies to the IL6R variants is carried out by SPRusing the ProteOn XPR36™ Array system (BioRad). An anti-human IgGsurface (Jackson Labs 109-005-008) was created on a GLC Biosensor chipby primary amine coupling. Test antibodies are captured on this surfaceas ligands. The IL6R variants are used as analytes and passed over thecaptured antibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Bindingcurves are double referenced using a buffer injection (i.e. OnM) toremove baseline drift and injection artefacts. Regeneration of thecapture surface is with 100 mM phosphoric acid which removes thecaptured antibody allowing another cycle of capture and binding. Thebinding sensorgrams generated are analysed using the 1:1 model inherentto the ProteOn XPR36 Array system analysis software. The assay isperformed at 25° C. and using 1×HBS-EP (Teknova) as running buffer.

References

The references cited herein are incorporated by reference in theirentirety

-   1. Ferreira et al (PLoS Genet. 2013 April; 9(4):e1003444. doi:    10.1371/journal.pgen.1003444. Epub 2013 Apr. 4, “Functional IL6R    358Ala allele impairs classical IL-6 receptor signaling [sic] and    influences risk of diverse in flammatory diseases”; Rantala A et al,    Hum Immunol. 2011 Janurary; 72(1):63-8. doi:    10.1016/j.humimm.2010.10.010. Epub 2010 Oct. 15, “Association of    IL-6 and IL-6R gene polymorphisms with susceptibility to respiratory    tract infections in young Finnish men”;-   2. Zhang H Y et al, Oral Dis. 2014 January; 20(1):69-75. doi:    10.1111/odi.12075. Epub 2013 Feb. 24, “The association of IL-6 and    IL-6R gene polymorphisms with chronic periodontitis in a Chinese    population”;-   3. J C Galicia et al, Genes and Immunity (2004) 5, 513-516.    doi:10.1038/sj.gene.6364120 Published online 12 Aug. 2004,    “Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence    that serum levels of soluble IL-6R are genetically influenced”;-   4. Esparza-Gordillo J et al, J Allergy Clin Immunol. 2013 August;    132(2):371-7. doi: 10.1016/j.jaci.2013.01.057. Epub 2013 Apr. 9, “A    functional IL-6 receptor (IL6R) variant is a risk factor for    persistent atopic dermatitis”.

TABLE 14 1000 GENOMES PROJECT HUMAN POPULATIONS Below is a summary ofthe ethnic populations as per the 1000 Genomes Project sequences. SuperPopulation Population Code Population Description Code (a) 100 GenomePopulations CHB Han Chinese in Bejing, China ASN JPT Japanese in Tokyo,Japan ASN CHS Southern Han Chinese ASN CDX Chinese Dai in Xishuangbanna,China ASN KHV Kinh in Ho Chi Minh City, Vietnam ASN CEU Utah Residents(CEPH) with Northern and EUR Western European ancestry TSI Toscani inItalia EUR FIN Finnish in Finland EUR GBR British in England andScotland EUR IBS Iberian population in Spain EUR YRI Yoruba in Ibadan,Nigera AFR LWK Luhya in Webuye, Kenya AFR GWD Gambian in WesternDivisons in The Gambia AFR MSL Mende in Sierra Leone AFR ESN Esan inNigera AFR ASW Americans of African Ancestry in SW USA AFR ACB AfricanCarribbeans in Barbados AFR MXL Mexican Ancestry from Los Angeles USAAMR PUR Puerto Ricans from Puerto Rico AMR CLM Colombians from Medellin,Colombia AMR PEL Peruvians from Lima, Peru AMR GIH Gujarati Indian fromHouston, Texas SAN PJL Punjabi from Lahore, Pakistan SAN BEB Bengalifrom Bangladesh SAN STU Sri Lankan Tamil from the UK SAN ITU IndianTelugu from the UK SAN (b) Super Populations AFR, African AMR, Ad MixedAmerican ASN, East Asian EUR, European SAN, South Asian (c) PopulationAncestries European ancestry Utah residents (CEPH) with Northern andWestern European ancestry (CEU) Toscani in Italia (TSI) British fromEnglandand Scotland (GBR) Finnish from Finland (FIN) Iberian populationsin Spam (IBS) East Asian ancestry Han Chinese in Beijing China (CHB)Japanese in Toyko, Japan (JPT) Han Chinese South (CHS) Chinese Dai inXishuangbanna (CDX) Kinh in Ho Chi Minh City, Vietnam (KHV) Chinese inDenver, Colorado (CHD) (pilot 3 only) West African ancestry Yoruba inIbadan, Nigeria (YRI) Luhya in Webuye, Kenya (LWK) Gambian in WesternDivision, The Gambia (GWD) Malawian in Blantyre, Malawi (MAB) WestAfrican Population (TBD) Americas African Ancestry in Southwest US (ASW)African American in Jackson, MS (AJM) African Caribbean in Barbados(ACB) Mexican Ancestry in Los Angeles, CA (MXL) Puerto Rican in PuertoRico (PUR) Colombian in Medellin, Colombia (CLM) Peruvian in Lima, Peru(PEL) South Asian ancestry Ahom in the State of Assam, India Kayadtha inCalcutta, India Reddy in Hyderabad, India Maratha in Bombay, IndiaPunjabi in Lahore, Pakistan

TABLE 15 Exemplary anti-IL6R disclosures, eg of antibodies and/orantibody fragments, assays, treatments, formulations, kits, methods andindications, useful in any and all aspects of the invention Patent orpatent application which is incorporated by reference in its entirety,and specifically, eg, with respect to the SEQ ID Nos. comprising ananti-IL6R monoclonal antibody or fragment thereof U.S. Pat. No.8,568,721, US20130157313A1, US20130149310A1, US20130122003A1, U.S. Pat.No. 8,192,741, U.S. Pat. No. 8,183,014, US20120003697A1, U.S. Pat. No.8,080,248, U.S. Pat. No. 8,043,617, US20110171241A1, US20100316636A1,US20100316627A1, U.S. Pat. No. 7,582,298 

TABLE 16 Human antibody gene segment variants distributed over severalhuman ethnic populations - useful for ligand tailoring ExampleNucleotide Hom Human Amino Acid Variation³ Freq⁸ Gene ExampleCoordinate² (NCBI dbSNP Variant (Het + Segment Human & referenceNucleotide Human No. Het Hom Cum Type Allele¹ Variation number)⁴Position Populations⁵ Individs⁶ Freq⁷ freq)⁹ Freq¹⁰ IGHV3-9 IGHV3-9*0130F TTT (IMGT numbering) (Phe at position 4 in SEQ ID NO: 32) (CDR1variation) IGHV3-9*02 30S TCT (IMGT numbering) (CDR1 variation)IGHV3-9*01 110T ACG 14:106552310 B 1015 0.082 0.847 0.888 (Thr atposition 33 in (forward strand) (0.929) SEQ ID NO: 34) (FW3 variation)IGHV3-9*03 110M ATG 14:106552310 B 167 0.082 0.071 0.112 (FW3 variation)(rs8020204) (forward strand) (0.153) IGKV3-11 IGKV3-11*01 115P CCT 2:89326669 B 1090 0.064 0.934 0.966 (Pro at position 7 in (forwardstrand) (0.998) SEQ ID NO: 36) (CDR3 variation) 115H CAT  2:89326669 B72 0.064 0.002 0.034 (CDR3 variation) (rs182958807) (forward strand)(0.066) IGKV3-11*01 87S TCT  2:89326754 B 1090 0.074 0.924 0.961 (Ser atposition 15 in (forward strand) (0.998) SEQ ID NO: 38) (FW3 variation)87P CCT  2:89326754 B 83 0.074 0.002 0.039 (FW3 variation) (rs191612627)(forward strand) (0.076) IGKC IGKC*01 84V GTC  2:89156948 B 454 0.3450.071 0.243 (Val at position 84 in (forward strand) (0.416) SEQ ID NO:16) IGKC*04 84L CTC  2:89156948 B 1015 0.345 0.584 0.757 (rs232230)(forward strand) (0.929) IGKC*01 87C TGC  2:89156939 (Cys at position 87in (forward strand) SEQ ID NO: 16) IGKC*02 87G GGC  2:89156939(rs200765148) (forward strand) IGHG1 IGHG1*01 204D GAT 14:106208086 A153 0.400 0.096 0.296 (CH3 variation) (forward strand) (European (0.496)ancestry) IGHG1*03 204E GAG 14:106208086 A 366 0.400 0.504 0.704 (CH3variation) (rs1045853) (forward strand) (European (0.904) ancestryIGHG1*01 206L CTG 14:106208082 A 0.358 0.104 0.283 (CH3 variation)(forward strand) (European (0.462) ancestry) IGHG1*03 206M ATG14:106208082 A 0.358 0.538 0.717 (CH3 variation) (rs11621259) (forwardstrand) (European (0.896) ancestry) IGHG2 IGHG2*01 72P CCC 14:106110914B 0.336 0.540 0.708 (CH1 variation) (forward strand) (0.876) IGHG2*0272T ACC 14:106110914 B 0.336 0.124 0.292 (CH1 variation) (rs11627594)(forward strand) IGHG2*01 75N AAC 14:106110904 A 0.007 0.993 0.997 (CH1variation) (forward strand) IGHG2*04 75S AGC 14:106110904 A 0.007 0.004(CH1 variation) (rs201590297) (forward strand) IGHG2*01 76F TTC (CH1variation) IGHG2*04 76L TTG (CH1 variation) IGHG2*01 161V GTG14:10611013 B 0.342 0.539 0.711 (CH2 variation) (forward strand) (0.881)IGHG2*02 161M ATG 14:10611013 B 0.342 0.118 0.289 (CH2 variation)(rs8009156) (forward strand) (0.46) IGHG2*01 257A GCC 14:106109752 C0.199 0.493 0.592 (CH3 variation) (forward strand) (0.692) D 0.007 0.9920.995 (0.999) IGHG2*06 257S TCC 14:106109752 C 0.199 0.308 0.408 (CH3variation) (rs4983499) (forward strand) (0.507) D 0.007 0.002 0.005(0.009) Table Footnotes: ¹⁰IMGT notation (ww.imgt.org); refer to figuresfor other alleles comprising this variation. ¹¹Numbering as indicated inEnsembl (available on the World Wide Web at ensembl.org) unlessotherwise indicated ¹²SNP Underlined in Codon. ¹³NCBI dbSNP Build 138released on Apr. 25, 2013. ¹⁴Human population used for representativevariant frequency analysis. Populations A This population included 662participants of European descent from the ClinSeq project, all of whomhad undergone whole-exome sequencing using Agilent's 38 Mb or 50 Mbcapture kit. B 1000 Genomes database. C ESP6500: African_American. DESP6500: European_American. ¹⁵Number of individuals in representativepopulation found to have the allele. ¹⁶Heterozygous human genotypefrequency, ie, cumulative frequency of all genotypes having oneoccurrence of the variant allele and one occurrence of another allele(heterozygous state), eg, ac genotype in the population. ¹⁷Homozygoushuman genotype frequency, ie, cumulative frequency of two occurrences ofthe variant allele (homozygous state), eg, cc genotype in thepopulation. ¹⁸Total human genotype frequency, ie, total of heterozygousplus homozygous human genotype frequencies. ¹⁹Cumulative human allelefrequency of all occurrences of the variant allele in the population.

TABLE 17 SEQUENCES SEQ ID NO: Human AlleleNucleotide/Amino Acid Sequence  78 IL-6RMLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQ D SSSVPLPTFLVAGGSLAFGT LLCIAI VLRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFPR D  = position 358 V  = position 385 79 ATGCTGGCCGTCGGCTGCGCGCTGCTGGCTGCCCTGCTGGCCGCGCCGGGAGCGGCGCTGGCCCCAAGGC GCTGCCCTGCGCAGGAGGTGGCGAGAGGCGTGCTGACCAGTCTGCCAGGAGACAGCGTGACTCTGACCTG CCCGGGGGTAGAGCCGGAAGACAATGCCACTGTTCACTGGGTGCTCAGGAAGCCGGCTGCAGGCTCCCAC CCCAGCAGATGGGCTGGCATGGGAAGGAGGCTGCTGCTGAGGTCGGTGCAGCTCCACGACTCTGGAAACT ATTCATGCTACCGGGCCGGCCGCCCAGCTGGGACTGTGCACTTGCTGGTGGATGTTCCCCCCGAGGAGCC CCAGCTCTCCTGCTTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGTGGGGTCCTCGGAGCACCCCA TCCCTGACGACAAAGGCTGTGCTCTTGGTGAGGAAGTTTCAGAACAGTCCGGCCGAAGACTTCCAGGAGC CGTGCCAGTATTCCCAGGAGTCCCAGAAGTTCTCCTGCCAGTTAGCAGTCCCGGAGGGAGACAGCTCTTT CTACATAGTGTCCATGTGCGTCGCCAGTAGTGTCGGGAGCAAGTTCAGCAAAACTCAAACCTTTCAGGGT TGTGGAATCTTGCAGCCTGATCCGCCTGCCAACATCACAGTCACTGCCGTGGCCAGAAACCCCCGCTGGC TCAGTGTCACCTGGCAAGACCCCCACTCCTGGAACTCATCTTTCTACAGACTACGGTTTGAGCTCAGATA TCGGGCTGAACGGTCAAAGACATTCACAACATGGATGGTCAAGGACCTCCAGCATCACTGTGTCATCCAC GACGCCTGGAGCGGCCTGAGGCACGTGGTGCAGCTTCGTGCCCAGGAGGAGTTCGGGCAAGGCGAGTGGA GCGAGTGGAGCCCGGAGGCCATGGGCACGCCTTGGACAGAATCCAGGAGTCCTCCAGCTGAGAACGAGGT GTCCACCCCCATGCAGGCACTTACTACTAATAAAGACGATGATAATATTCTCTTCAGAGATTCTGCAAAT GCGACAAGCCTCCCAGTGCAAGATTCTTCTTCAGTACCACTGCCCACATTCCTGGTTGCTGGAGGGAGCC TGGCCTTCGGAACGCTCCTCTGCATTGCCATTGTTCTGAGGTTCAAGAAGACGTGGAAGCTGCGGGCTCT GAAGGAAGGCAAGACAAGCATGCATCCGCCGTACTCTTTGGGGCAGCTGGTCCCGGAGAGGCCTCGACCC ACCCCAGTGCTTGTTCCTCTCATCTCCCCACCGGTGTCCCCCAGCAGCCTGGGGTCTGACAATACCTCGA GCCACAACCGACCAGATGCCAGGGACCCACGGAGCCCTTATGACATCAGCAATACAGACTACTTCTTCCC CAGATAG HEAVY CHAIN ALLELES  80 IGHG1*01gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctggg (CH1 +Hinge + ggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgCH2 + CH3 + tggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaCH − S) ggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa  81ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR D E LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK D  = position 204 L  = position 206  82IGHG2*01 gcctccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgag(CH1 + Hinge +agcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcg CH2 + CH3 +tggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctca CH − S)ggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa  83ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVP SS NF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG V EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI A VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK P  = position 72 N  = position 75 F  =position 76 V  = position 161 A  = position 257  84 IGHV3-9*01gaagtgcagctggtggagtctgggggaggcttggtacagcctggcaggtccctgagactctcctgtgcagcctctggattcacct t tgatgattatgccatgcactgggtccggcaagctccagggaagggcctggagtgggtctcaggtattagttggaatagtggtagcataggctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctgagagctgaggac a cggccttgtattactgtgcaaaagata t  = nucleotide number 86 c  =nucleotide number 272  85 EVQLVESGGGLVQPGRSLRLSCAASGFT FDDYAMHWVRQAPGKGLEWVSGISWN SGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAED TALYYCAKD F  = position 30 (IMGT nomenclature) T  =position 110 (dbSNP nomenclature)  86 IGHV3-7*01gaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctg agactctcctgtgcagcctctggattcacctttagtagctattggatgagctgggtccgc caggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaa tactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactca ctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaga ga  87EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR  88 IGHJ3*02T GAT GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA G  89D   A   F   D   I   W   G   Q   G   T   M   V   T   V   S S  90 IGHJ6*01AT TAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGG CAA GGGACC ACG GTC ACC GTC TCC TCA G  91Y   Y   Y   Y   Y   G   M   D   V   W   G   Q   G   TT   V   T   V   S   S LIGHT CHAIN ALLELES  92 IGKC*01cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt  93RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK V YA C EVTHQGLSSPVTKSFNRGEC V  = position 84 C = position 87  94 IGLC1*01cccaaggccaaccccacggtcactctgttcccgccctcctctgaggagctccaagccaacaaggccacactagtgtgtctgatcagtgacttctacccgggagctgtgacagtggcttggaaggcagatggcagccccgtcaaggcgggagtggagacgaccaaaccctccaaacagagcaacaacaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca  95 PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS  96 IGKV1-12*01gacatccagatgacccagtctccatcttccgtgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttactattgtcaacaggctaacagtttccctcc  97DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP  98 IGKV3-11*01gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtggg t ctgggacagacttcactotcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggc c tcc t  =nucleotide number 199 c  = nucleotide number 284  99EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSG SGTDFTLTISSLEPEDFAVYYCQQRSNW P S  = position 87 P  = position 115 100IGKJ2*01 tgtacacttttggccaggggaccaagctggagatcaaac 101 YTFGQGTKLEIK 102IGKJ4*01 G CTC ACT TTC GGC GGA GGG ACC AAG GTG GAG ATC AAA C 103L   T   F   G   G   G   T   K   V   E   I   K 104 An IGHG1*01EVQLVESGGG LVQPGRSLRL SCAASRFTFD DYAMHWVRQA PGKGLEWVSG Heavy ChainISWNSGRIGY ADSVKGRFTI SRDNAENSLF LQMNGLRAED TALYYCAKGR DSFDIWGQGTMVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFPAVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPAPELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKPREEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVTYLPPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 105 An IGKC*01DIQMTQSPSS VSASVGDRVT ITCRASQGIS SWLAWYQQKP GKAPKLLIYG Kappa LightASSLESGVPS Chain RFSGSGSGTD FTLTISSLQP EDFASYYCQQ ANSFPYTFGQ GTKLEIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 106 An IGHG1*01EVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAKISPG Heavy ChainGSWTYY SDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYALDIWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 107 An IGKC*01EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLA Kappa LightSGIPAR Chain FSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 108 IGHV3-9*01 ggattcacct ttgatgattatgcc CDR1 (IMGT t  = position 11 109 nomenclature) G   F   T   F    D   D   Y   A F  = position 4 110 IGHV3-9*01ggc tat gcg gac tct gtg aag ggc cga ttc acc atc tcc FW3 (IMGTaga gac aac gcc aag aac tcc ctg tat ctg caa atg aac nomenclature)agt ctg aga gct gag gac a c g gcc ttg tat tac tgt c  = position 98 111G   Y   A   D   S   V   K   G   R   F   T   I   S   RD   N   A   K   N   S   L  Y   L   Q   M   N   S   L R   A   E   D    T   A  L  Y   Y   C T  = position 33 112 IGKV3-11*01CAG CAG CGT AGC AAC TGG C C T CC CDR3 (IMGT C  = nucleotide 20 113Nomencalture) QQRSNW P P  = position 7 114 IGKV3-11*01aacagggccactggcatcccagccaggttcagtggcagtggg t ctgggacagacttcact FW3 (IMGTctcaccatcagcagcctagagcctgaagattttgcagtttattactgt Nomencalture) T  =nucleotide 43 115 NRATGIPARFSGSG S GTDFTLTISSLEPEDFAVYYC S  =position 15 127 AfliberceptSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDS polypeptideRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTII

sequence

FVRVHEKDKTHTCPPCP A PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA K

QPREPQVYTLPPSR D E L TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 127) DKTHTCPPCP (SEQ ID NO: 128) =C-terminal part of IGHG1*01 hinge A  =start of human IGHG1*01 CH2 sequence K  =end of human IGHG1*01 CH2 sequence

 = start of human IGHG1*01 CH3 sequence D  =Asp204 (corresponding to position 204 in SEQ ID NO: 42) L  =Leu 206 (corresponding to position 206 in SEQ ID NO: 42)Flt1 Ig-like C2-type 2 domainGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGH (SEQ ID NO: 129) KDR Ig-like C2-type 3 domain Y

 (SEQ ID NO: 130) 129 Flt1 Ig-likeGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEAT C2-type 2VNGH domain 130 KDR Ig-likeYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGS C2-type 3EMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNST domain 131 A human VEGFAMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD sequenceIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM(isoform 206)SFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR 132 A human VEGFAMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD sequenceIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEM(isoform 165)SFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELN ERTCRCDKPRR133 A human PD-L1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIsequence VYWEME (ENST00000381577)DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRC MISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQV LSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHP PNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET 134 A human IL-2MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL sequenceTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 135 Nav1.8 AMINOMEFPIGSLETNNFRRFTPESLVEIEKQIAAKQGTKKAREKHREQKDQEEKPRPQLDLKACACID SEQUENCENQLPKFYGELPAELIGEPLEDLDPFYSTHRTFMVLNKGRTISRFSATRALWLFSPFNLIRRTAIKVSVHSWFSLFITVTILVNCVCMTRTDLPEKIEYVFTVIYTFEALIKILARGFCLNEFTYLRDPWNWLDFSVITLAYVGTAIDLRGISGLRTFRVLRALKTVSVIPGLKVIVGALIHSVKKLADVTILTIFCLSVFALVGLQLFKGNLKNKCVKNDMAVNETTNYSSHRKPDIYINKRGTSDPLLCGNGSDSGHCPDGYICLKTSDNPDFNYTSFDSFAWAFLSLFRLMTQDSWERLYQQTLRTSGKIYMIFFVLVIFLGSFYLVNLILAVVTMAYEEQNQATTDEIEAKEKKFQEALEMLRKEQEVLAALGIDTTSLHSHNGSPLTSKNASERRHRIKPRVSEGSTEDNKSPRSDPYNQRRMSFLGLASGKRRASHGSVFHFRSPGRDISLPEGVTDDGVFPGDHESHRGSLLLGGGAGQQGPLPRSPLPQPSNPDSRHGEDEHQPPPTSELAPGAVDVSAFDAGQKKTFLSAEYLDEPFRAQRAMSVVSIITSVLEELEESEQKCPPCLTSLSQKYLIWDCCPMWVKLKTILFGLVTDPFAELTITLCIVVNTIFMAMEHHGMSPTFEAMLQIGNIVFTIFFTAEMVFKIIAFDPYYYFQKKWNIFDCIIVTVSLLELGVAKKGSLSVLRSFRLLRVFKLAKSWPTLNTLIKIIGNSVGALGNLTIILAIIVFVFALVGKQLLGENYRNNRKNISAPHEDWPRWHMHDFFHSFLIVFRILCGEWIENMWACMEVGQKSICLILFLTVMVLGNLVVLNLFIALLLNSFSADNLTAPEDDGEVNNLQVALARIQVFGHRTKQALCSFFSRSCPFPQPKAEPELVVKLPLSSSKAENHIAANTARGSSGGLQAPRGPRDEHSDFIANPTVWVSVPIAEGESDLDDLEDDGGEDAQSFQQEVIPKGQQEQLQQVERCGDHLTPRSPGTGTSSEDLAPSLGETWKDESVPQVPAEGVDDTSSSEGSTVDCLDPEEILRKIPELADDLEEPDDCFTEGCIRHCPCCKLDTTKSPWDVGWQVRKTCYRIVEHSWFESFIIFMILLSSGSLAFEDYYLDQKPTVKALLEYTDRVFTFIFVFEMLLKWVAYGFKKYFTNAWCWLDFLIVNISLISLTAKILEYSEVAPIKALRTLRALRPLRALSRFEGMRVVVDALVGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGKFWRCINYTDGEFSLVPLSIVNNKSDCKIQNSTGSFFWVNVKVNFDNVAMGYLALLQVATFKGWMDIMYAAVDSREVNMQPKWEDNVYMYLYFVIFIIFGGFFTLNLFVGVIIDNFNQQKKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKFQGFVFDIVTRQAFDITIMVLICLNMITMMVETDDQSEEKTKILGKINQFFVAVFTGECVMKMFALRQYYFTNGWNVFDFIVVVLSIASLIFSAILKSLQSYFSPTLFRVIRLARIGRILRLIRAAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYSIFGMSSFPHVRWEAGIDDMFNFQTFANSMLCLFQITTSAGWDGLLSPILNTGPPYCDPNLPNSNGTRGDCGSPAVGIIFFTTYIIISFLIMVNMYIAVILENFNVATEESTEPLSEDDFDMFYETWEKFDPEATQFITFSALSDFADTLSGPLRIPKPNRNILIQMDLPLVPGDKIHCLDILFAFTKNVLGESGELDSLKANMEEKEMATNLSKSSYEPIATTLRWKQEDISATVIQKAYRSYVLHRSMALSNTPCVPRAEEEAASLPDEGFVAFTANENCVLPDKSETASATSFPPSYESVTRGLSDRVNMRTSSSIQNEDEATSMELIAPGP 136 Nav1.8AAAAGTGACTGGAAACAGTGAGTAGAAGTAAGTCCTTTATTAAAAGCCAATAGAGAACAG NUCLEOTIDEGAGAGAGGAGGGAAGACCTGGATGGGCTCTGCATCAAGGGAAAGTTTTTAAAGTTGGAGG SEQUENCEAAAGTTAAGCATCTTTAAATGCTGATAGGAAAGAGCTGGCAGACAAGGAGAGAGAAAAAAATGAGGTAGATATGAGATAAGATCCTAAGCAGAGGGAAAGGAATGGTCCTTAGAATGTAGGAAGATACCTCTCATGTTGTAAGAGAAGGGAAGGAGGAAAGAAGGCTGCAGACACAGGTGAGTTGGAGGTTTGATGGTATGAAGTTGGCAGAGTTCTGTTTTTCTCCATGAACTAGGAAATATGGTCATTGGCTTACTGCAAGGGAAGACAGGGAATAGTGAGAAAAGAGTGTATTTGAAACCTCAGACTAGAAGAAACTGCTGACTAGGGATACCATATGATTCCCAGGCAATACTGAGGTTCCCCATAACCCCTCAGCAGTTCAGGGGAACTGTTCTAGATTGACTTAGCCTTGGGATTTAGGATAAGGGTGAGGGAGTGGCAAAATTGTGGACCACAGGTCTAATCTGGTTATGGGGAAAAGTGTTCAGCCACAGACTGAACCTGGAGGTCCCAGAGAAATTGGAGGTCCCAATGAAATTCTCCAATAATCTGGAGTTCTCCAGTAACTTGGAGGTCCAAATGAAATTCAAACAGGTGGAGAAATGTTGACAATGTCAATAAATAAAAGATTGTGATAAGAAAGTAGCATGAGACTTAAGATAAAGTAGACTATAAACCAAAGTCTTTGATGAATATGGGAGAGTGCCTGGAACTCAGTGGTCCTGGAAATGAGGAGAGAGGGCAGCATGGCTAAATCTGGTGGTGGTGGTAGGATGGTACAAAGTGGTGAAGAAGCAATGACATAGAAGCACATGGGGAGCAAAGACATAACCCTGAGGCACCTGGAGGAGGGTTGAATAAACAGCCTCCCCTGAATGCCTTGCAGGGGAATGGGTTCCTGGGAGGAGCCAAGTGTGAGTTCAGGGAGGGGCCAGATATGAGGGTGGGAGAAGGGCTGTTCTGACAATCAAGATGGGACAGATGAGAGGGACAGGGCCAGTGGGCAAGCTGTCACCTCTCTGTGGTTATGTCCACTCTTATAAGAGTATAAATACTTCCTGAAGAAGAATGAGAAGATGGAATTCCCCATTGGATCCCTCGAAACTAACAACTTCCGTCGCTTTACTCCGGAGTCACTGGTGGAGATAGAGAAGCAAATTGCTGCCAAGCAGGGAACAAAGAAAGCCAGAGAGAAGCATAGGGAGCAGAAGGACCAAGAAGAGAAGCCTCGGCCCCAGCTGGACTTGAAAGCCTGCAACCAGCTGCCCAAGTTCTATGGTGAGCTCCCAGCAGAACTGATCGGGGAGCCCCTGGAGGATCTAGATCCGTTCTACAGCACACACCGGGTAAGAGCTACTAGTAGGAATGTCTGCTCTCAGCTCTTGGAGAGGAAGTCTCGGTCCCACCCAGCCCAGAAAGTCCTCCTTGGCCCTGGGTGATGTGGCAGTCTGCTAAAGATTCCGTTAACAACCAAAAAAAATATATATATATATCTCTAACTTATTTGATGATCTCTTTGAATTTTTATGACATTTATGATAGAAACTAACTAAACCCACATCTTCAAAAGCAGATAACTGCTGCAGAGCTTTCAGGGAAAGTTGGCTGTTCCCTTGGATGACCTCTGGCCTGGGGCCTCCAGAGGAGTTGGGTCTCTTTGCAGGTGGAGAGCAGACATCTTGAGGTTGGGGGAGGAGCATGGCGGTGAGGACAGAAAGCAGGATGAATTTAAGCCAGTGGTCCTCAACTATGATCTTGGAACCCCTGGTGTCCCCAAAACTGTTTTAGTGGGTTTATAAAATGAAAACTATTGTATAACAATACTTGGATGTAATTTGTCTTTTTCACACACTTTCTCTTATGATCATACATTGGTGAATATAGAGGCTATTGATGGGGGATGATGTCATCACTCTAAAAGCTACTGGAATGTGTGCCTGTATATTCCCGTGTTTTAAAATTTCCTGAGTTTTGAATTTCTAATATGAGATATATATACACACACATATGATGTATATATGTGTGTATACATCTATACACTATATATATACATACAACTATGTATATACATACAACTAGACCATGTAGCTAGTTATGTATATATAGGTATATAAGCATATATACATATATATTAATTTCTAATATGATATACATATATCTGGCATAAACAAAAAGGTATCAACATAAACAAAAGTTATTTGGGGTCCTCAATAAGGTTTGAGAGTAATAATCCCTGAAATCAAAAAGTTTGAGAATCAGTAGTTTAAACAAGAGCTTCTTCATGACAATTATCTCACCTGTGTTGTATTTTTGATGTTCTACGAGCCAAAAAATGGTGAATGCATGTTTGGGAGTTAGGAGGACAGGAATCAGCCATCTCAAATATGGCTAGCAGAGTAAATAAAAAGGCACTGAATATCTTTCTTCCTCCTCTCCCATGCCAAGAGTTCATATCCTCCTAAATCCTCAGAGGGAGAAACCAGGAGAGGATTTAAGAGACAGAGATGTAGAAGGTGGAGACAGAAGAAACATTACAAAACAAGGATGGTTCTCTTGTCAGAAGAGAAATTAAATGCAACCATAACTTCACCATCCTGTTGCCAGAGTCCTCCCCTGGGCATAACACCTCTTACCAGCCTGAGGATTGGCTGTCTGTCCATTAAAGTACCCTCAGTGGGCTCATCTTGGTGCACTGTCAGTCCCAGAGATGAGGATCTTGCTGAGAGGGTGAGGATATGGCTTTTAGTCCTTGATCCTCTCTGCCTCTCTGTACCTGCATTTGACTCTCATTGACCTGTAGTTCCCAGAGCCCTTCTTGCTCATAAGCCTGAGATAATGCCTCTCATGTTTAAAAACATGGTTGTATAAGGAATCTTGTTTCTCATTGGAGGTCCACTTTCTGTTTTGTTTTGTTTCAGACATTTATGGTGCTGAACAAAGGGAGGACCATTTCCCGGTTTAGTGCCACTCGGGCCCTGTGGCTATTCAGTCCTTTCAACCTGATCAGAAGAACGGCCATCAAAGTGTCTGTCCACTCATATCCTTTGCACGATTGCCTCTTCCACGTGTTACAATTAGACCTTGCCTACTGTGTCCAGTACCTTCTTTAGAGGTTAGCAACAGAGCCCTTATCCAATTGAATCCCAAAAGTACAATGAGGTCATTAGATTGAAAAAATGGCTTGGGCCTTTAATGTGTCCCACACCCCAAAGCAGTCCCAAGACACAGAAGAAGTGTTTATGCAACACACAGGTGGTTTGGATTCACCTCAGACTAATCTAGACACACCTGTGGATGCACAACTTGAAAAACATCCTCTTTATGAGGGGAGGTTTGCTGATGCCCCCAGACACATTCTCACCACGGTGTGTGGTCCGCTTTCCCCACAACACAACAGGCAGTAAGAGGGCCCAAGCCTTCTTTGGGCAGATGAGATAGGCATGAGTTGTTGCTCTGTGCCATAGCCATGCTGAGAAGTGGCTGCTTACTGGGCAAAAGAGCAGCACCTCCCAGAGGGCAAGGAAAACAGACCATGATGGGAAGAGCACACATAGTCTCAATGGGACCTCATTGGTCATGGGGCACTGTTAACATCACAAATTACTCAAAAACTACTAGCAGGTTTAAAATGTCAATAATCTGATGTTCATGCCATACAAGACTGTAATGATTAGAGACACATTGGTCAAATGTCTACTATATGAATTGTTTTAGGTAATGTCAGAAGCTGCTTTCAGGGGATTTTGCAAGGCAAGCCAAACTAGTAGAAAAAAGATACTTTGGTTTGGTTTCCTAAGATCTTCCTGGGTGATCCTAATGGAAAGATCCTAATGACCTAAGATCCTTGGTCATCGGAAGGATTGGAAGTTCTTTATGACAAATTTAGCCCTGGCTTGCAGGAGGTTGATGTACAAGAATACACCTGCCTCAGTTGCCTGACAAATGGTAACCAGCCTTTTCTTAGAAGGCTTCAGTAAAAAAGAGTTCTACCCTGCTCCCTAACTCTGAGGCAGATTGTTGGATTTCTGGACAGTTGTCATTGAGCTGGTAACCTTGGCACAGTCATCTATCTGCACACTCAACATGAAGCTGCACAGCTTGATGAAGTTGCATTACGTTTTCTCTTTTTACATTTTTTTCTTTCTGGCTGGCCAGCAAGAATTACGTTTTCTGAGCTGAATCTTGAATTTTCAGATTCTACAGAAGGATATTTAATGACAATACAGTCTCTCACTTGCTAAATACTTCCTAAGTGTCAATACCATCATCAGTTCAAAAGCCTCTGAAGTAGAGATTACCATCCCCATTTTACAGATAATGAAACTGACACACAGAGGCATTAAGTAAATTATCCAAGACCATGTAGCTAGTTAAGAAGTCCAGCCTGCATCCTGCGTAGAGCAGTATCTACAGTGAGGAAGTAGGGTGGAAAGTAATCAGGGTAATTATTCTCTCAATGTGTCGTCTCTGCTTGGGTAGTTGTTGCTTTTTCAAAATATATACAATTACATGGAAATTACAAATAAAGAGAAATCACAAACATACAAACTCCTCTTTATGGATTCTGGTTGAATTACATCTGGATAAGTAAGAAACAGCTAATTAGAAATAAAATCCCCTAAAAAGAACCAAGTGACCAGTGAATATTCTCTTCATCTTGAGATTTGATGAGTAAATAATTGCTTGGTAGAGTCACAGCTGGACAGAAAAGCTGTTGAGGGGGTCTCTTCCTCACAAAAGTCTGCATTTAAATCTCATAAATTTGGTAACTCCAGCCCCAAAAGATACTAGAATTCTCAGGGCCCTATTATTAATATTTCACCAAAAATATAATTCTAATCACATTTGCTATGAATGTTTTCATAACTAATATTATACATAAGATATATTACTTTTAAATATTTGTAGAATTGGAGACACGATTTTAGTTTAATCCTTGGATTAACATTTCCCCCCATGTGAGCATATTTCTTAATTTAATTGTAAGTCAGCAGGAAAATGTGGTTTAATTAATAGAATTTTCCTTTTCATAATAGATGTGTTCCTAAGTAACTGTAAAACTAGACACATCAAGGCATTTACAGAAAACTAAAATACATTTAAATACTTCTGCTCAAATTGAAAAATGAGTGTTGTACTCTCTCTGTAGGTCAAGTTTATCTTCAAGTTTTATTTTTCCTTAGGAAGCACTGTTGGGAGCCCAGCTATTCCTAAGAAAAATGTCCAAGTGGGAACAATTTACCTTTAGATTCTCCCCACCAACATTTCTTCCCTTCTATTTTCCCCTTCCACCCACCTTCCTTTCAATGTCTCCCTCCCCTTTTGATAATCCCTTTGCCCATAGTCTGCTGTCAATATTCTCCACTCTAGCTATTCAGAGTTCTCCACTCCAGCCTAATCCTACTTGGGGTTTTCTTCCTGAGCCAGTATTTCATTTCCCTGTGCCTGCCTCCACCGTTCCTACCCAGAAGACCATTTCCTCGGTGGAACTGATCTCAACTAAGTTACCCAAACAATCTGATGAAACTAATTCCCATGAAAGTTTGTACCTCTTCACTTATCTCCAAGTTTCTAACTTTTCTTTGTCTTTTAGAATAAAAAAGACATTTGCCAGATGAAATTTCAGGCCATTTTGTATACTGAAATGAAGGCTTCGGGGCAGAAGGACATTTTGGAAAAGGGAGTCTGTTTCCTTCTATCTTGCTCAGAGTTTGTTATCAAACAGATAAGAGTTCACATCTCTGCAGCATGGTTGACTAGCTGCATGACCTGAGAAAAGTTATTTAACTTTTATTTTTTGTCAGCCTGTTTCTCAGCTTCAAAATGTAGATATTTATACCCACCTGTAGATCAGTTGTATTAGAAGTAACATGGACAAAGTACTCAGCCTAGCTTAGGCACTCAGTAAATGTTAGTGGTTATTATGTGACAAGATCATAAAGCTAATATTGGCTAAGATTTATGAATGTAATCATTCAGCATCAAGGTGATCCTAGATGACATCACAGAGACTTGGTGATCTCAGCTTTTCCTTAACACAGGCCTTTCAGGTGGTTCAGTTTATTTATTACGGTCACTATTTTGGTTAATTGTGTGTGCATGACCCGAACTGACCTTCCAGAGAAAATTGAGTGAGTAGATTTGCTATCATTATTGTACCATCTCTTTTTGCTTTCTTTTCTTCCTTCACTTGGCAGTCTTTGTCTTTTATTTATCCTGCCATGCCTCATGTGGGTAATGCTGAATCTCATTGTCTATATGTCTTTGTAATAACCAGGTCTCTCCTTCTGTGAGATTTGTTTCATAAACATACATGATTTTCAGTTTCAAAGAGATACAGATTAGTGGTTCTCAACTTTGGCTGCATATTGCAATTTTTGGGGAGATTTTTTAAATGCCTGAGTCCCAGCCCCAGAAAGTCTAAGGTGGCCTGGGAAGATGAGTCTCCTCGCCCCCACCCCCCCCCAACACACACACACTCACTCACCTGCTAGATGCACACACAAATGTATGCCTTAGAGAAGGTATGCTTTTGAGAAGGCCTGCTATGAAAGAGAGACAGGAGCAAAACTGAGACAGAACTAAGAACAAATGGCTCTGGAGTATATTTAGAAAATAACTCATAAATGAAGCTGTTTAAGTTATCATGCTTCATATGCCTCAAAGTAACCTTAGTATTCCTGAAGTTATTCTATTTCCCTAGGTTAGTGGGTGAAACCCCTATAGTTTCAGAATCTGGGAGTTTTCCAAAAACATTCCATATCCACACATCTGGGAGGGATGCTTCCACTCCAGCTTGAACCTAACATACCTTTGGTTATAATAACAGGTGCTTTGCAGAGCTTTCTTTTTTTTTTTTTTTTTTTTTTTGCTAATCATAACAGTAATGCATGCCACAACAGAAAATACGTTATATTTTTATTAATAGATTAAAGGAAAAGGATCACATAGTCTTCTTAATTGATGCTAAAATCATTTGATAAAGTTTAATTTCCATTTATAATTTTAAATTCTTAGAAAAGTTGAAATAGAAGGGAATAAATGTGGCACATATACACCATGGAATGCTATGCAGTCATAAAAAAGGATGAGTTCATGTCCTTTGCAGGGACATGGATGAAGCTGGAAACCATCATTCTCAGCAAACTAACACAAGAACAGAAAACCAAACACCGTATGTTCTCACTCATAAGTGGGAGTTGAACAATGAGAACACATGGACACAGGGAGGGGAACACACTGGGGCCTGTTGAGGGGTGGGGGGCTAGGGGAGGGATAGCATTAGGAGAAATACCTAATGTAAATGACGGGTTGAAGGGTGCAGCAAACCACCATGGCACGGGTACACCTATGTAACAAACCGGCACGTTCTGCACATGTACCCCAGAACTTAAAGTATAATAATTTTAAAAAGGGGATTGCATCACTCTGATAAAGGGTATTCATTGTTTTAAAGTATCAAGCGTCATACTTAATTGTGAACATGGAAAGCTTTCCCTTTGAGATCAAAAGAAAAAAAAACCAAAGATGTCTGTTCTTCTCAGTTCCACTTTGTAAAGGAGGTCTCAGCCAGCATAGTAAAGCAAAGAAAAAAAAAGTGCAAGAAGAAAAAAGTACCGAAGATGTAATTTACAGATAATAAGATTGTATACATAAAATATCCAACATAACCTAGAGATAAACAGTAAGAATTAATAAAAGAGTATAGTAAGGTAGCTGGGTACAAATTAACATACATAAATCAATTGTGTATAGACACTAGCAACAAACAGAAAATAGTTTTTAAAAATACCATTTACACTGGCATTTTAAAATGTCAAGTATCTAGGAATAAGATTATCAAAGTATAAGTGAGATCTCTATAAGGAAAATTATAATAAGTATTGTGTAAAGTTTTTAAAGAAAACCAAATCAGTGGGGAGACATGTTCTCAGATTCAAAGTCTCAATATTGTAAAGACTCCCAAAAATGATTAATAAATTTAATATGATCCTCAAATCAATATTTCAACAGGCTTCTTTTGGTATTACTTGACAAACTTTAAACTTTAAATTTTCTTCCTCAAAAGTTTACATGAAATTTTAAAGAGCAAATTATAGCCAAGAAACTTTTGAGGAAGAAATACAAAGTGGGAGAACTCATTCTACTGGATGTCGAGATTTACTATAAAGCTCTAATTGAAGCAGTGTGCCATTGGTGTAAGATTGGTCAGAATAATGTAACAGCATAGACAGTCCATAAACAGACCAACACATATGTAAACATCTAATGTGTGAAAAGGTGGCACCCCAGAACAGCAGGGAAAAGGCGGTCTTCAAAAAATGATGCTGAAGAATTAGGAATCTCACTGGAAAAACTACATTAAACATCCAACTTTGCACCACACACAAGAATCAATTCTAGGTGGATCAACACCTCACTTGCTTTGTGAAATGCAAATATATAAAGCCCTTTTTTAAATTTTACTTTAAGTTCTGGGGTACATGTGCAGAAAGTGCAGGTTTGTTACATAGGTATACACATGTCATGGTGGTTTGCTGCACCCATCAACCCATCATCTACATTAGGTATTTCTCCTAATGCTATCCCTCCCCTAGCCCCCAACCCCTCAACAGGCCCTGGTGTGTGATGTCCCCCTCCCTGTATCCACGTGCTCTCATTGTTCATCTCCCACCTATGAGTGAGAACATGCGGTGTTTGGTTTTCTGTTCTTATGTCAGTTTGCTGAGAATGATGGTTTCCAGCTTCATCCATGTCCCTGCAAAGAACATGAACTCACCCTTTTTTATGGCTGCATAGTATTCCATGGTGTATATGTGCCATATTTTCTTTATCCAGTCTATCACTGATGGGCGTTTGGGTTAGTTCCAAGTCTTTGCTATTGTCAATAGTGCCACAATAAACATACGCATGCTTGTATCTTTATAGTAGAATAATTTATAATCCTTTGGGTATATACCCAGTAATGGAATTGCTGGGTCAAATGGTGTTTCTGGTTCTAGATCCTTGAGGAATCACCACACTGTCTTCCACAATGGTTGAACTAATTTACACTCCCACCAACAGTGTAAAAGCATTCCTATCTCTCCACATCCTCTCCAGCATCTGTTGTTTCCTGACTTTTTAATGATCTCCATTCTAACTGGTATGAGACTCCATCTCATTGTGGTTTTGACTGGCATTTCTCTAATGACCAGTGATGATGAGCTTTTTATCATATGTTTATTGGCTGCATAAATGTCTTCTTTTGAGAAGTGTCTGTTCATATCCTTTGCCCACTTTTTGATGGAGTTGTTTTTTCTTGTAAATTTGTTTAAGTTTTTTGTAGATTCTGGATATTAGCCCTTTGTCAGATTGATAGATTGCAAAAATTTTCTCCCATTCTGTAGGTTACCTATTCACTCTGATGATAGTTTCTTTTGCTGTGCAGAAGCTCTTTAGTTTAATTGGATTCCATTTGTCAATTTTGGCTTTTGTCACCATTGCTTTTGATGTGTTAGTCATAAAGCCTTTGCCCATGCCTATGTCCTGAAGGGTATTGCCTAGGTTTTCTTCTAGGGTTTTTATGGTTTTAGGTCTTATGTTTAAGTCTTTAATCTATCTTGAGTTAATTTTTGTGTAAGGTATAAGGAAGGGATCTAGTTTCAATTTTCTGCCTAAGGCTAGCCAGTTTTCCCCACACCATTTATTAAATAGGGAATCCTTTCCCCATTGCTTGCTTTTGTCAAGTTTGTCAAAGATCAGATGGTTGTAGATATGCGGTGTTACTTCTGAGGCCTCTGTTCTGTTCCATTGGTCTAGATATCTGTTTTGGTACCAGTACCTCGCTGTTTTGGTTACTGTAGCTTTGTAGTATAGCTTGAAGTCAGGTAGCGTGATGCCTCCAACTTTCTTCTTTTTGCTTAGAATTGTCTTGACTATGCGGGCTCTTTTTTGTTCCACATGAAATTTAAAGTAGATTTTTCTAATTCTGTGAAGAAAGTCAGTGGTAGCTTGATGGGGATAACATTGAATCTATAAATTACTTTGGGCAGTATGGCCATTTTCACAATATTGATTTTTCCTATCCATGAGCATGGAATGTTTTTCCATTTATTTGTGTCCTCTCTTATTTCCTTGAGCAGTGATTTTTAGTTCTCCTTGAAGAGGTCCTTCACATCCCTTGTAAGTTGTATTTCTAGATATTTTATTCTCTTTGTTGCAATTGTGAATGGGAGTTCACTCATGATTTGGCTCTCGGTTTGTCTGTTATTGGTGTATGGGAATGCTTGTGATTTTTGCACATTGATTTTGTATCCTGAGACTTTGCTGAAGTTGCTTATCAGCTTAAGGAGATTTTGGGCTGAGATGATGGGGTTTTCTAAATATACAGTCATGTCATCTGCAAACAGAGACAATTTGACTTCCTCTTTTCCTATTTGAATACCCCTTATTTCTTTCTCTTGCCTGATCGCCCTGGCCAGAACTTCCAATACTATGTTGAATAAGAGTGGTGAGATAGGGCATTCTTGTCTTGTGCTGGTTTTCAAAGGAAATGCTTCCAGTTTTTGCCCATTCAGTATGATATTGGCTGGGGGCTTGTCATAAATAGTTCTTATTATTTTGAGATACATTCCATCAATACCTAGTTTATTGAGAGCTTTTAGCATGAATGGGTGTTGAATTTTGTCGAAGACCTTTTCTGCATCTATTGAGATAATCACGTGGTTTTTGTCATTGGTTCTGTTTGTGTGATGGATTACGTTTATTGACTTGCGTATGTTGAACCAGCTTTGATCCCAGGTATGAAGCCGACTTGATCGTGGTGGATAAGCTTTTTGAAAGGAAAAAACAAAGAGGAATATCTTTATGACCTCAGACTAAGAAAAACTTTATTACATATGACACAAGAAGCACTAACTATACAGGAAAAAACTGATACATTTTAGTATACTATAATTAAGAATGTTCATCAGAAGACAACTTTAAGAGCATGAAAAAGCAAGTGCTTATATTCAGAGTAATGAACTCTACAAATCAATTAGAAAAATGTCAGAACAACCACTTGTAAAATAGCACAAAATTTGAATAGGCACTTTACAAAAGTGGAAATCCAAATGGCCATAAACATATGAAAGTATTCTTAACCTTTTTTTAAATCAAAGAGTTGCAAATTAAAATCACACTGAGATTTTATTACATGTTCATCATAGAAGGGAAATTTTTTAAAGTGTCACAATACCAAGTATTGGGAATGACATGGAGCAACAGAAACTCACACACTGCTGGTGGGAGTGTAAATTTGGACAACCTCTTTGGAAAGCAAGGGGTATTATCTAGTAAAGGTGTAAGCATACATATGCTGTATTCTTCTATTACACCCTTAGGTACCTATGTTTAACAGAAATATGTACTTGTGGACACCAGATTACAGCTACAAGAAAGTTTATGAACATTTGCCCATAAAAACCCCAAACCGGAAACCACTCAAATGTCCATAAACTTTGAGTACATTAACTATTTCTGTTATATTCTAAAATGAGAAAACTTACAGCAATAAAAATTAATAAACTTTAGTGCTGCACAACAATAAGGATGAGTCTTAAGAAAGTAATATTGAACAAAAAAAGCCAAATGCAAAGGATTACATAAACAAGGTTCATTTATATAAAGTTTGAAATCAAGCAAAACTAAACTATTTTGTTTAGGAATGCATACTTATGAAGTAAAAAGCATCAATAAAGTAAGGCAAGAAAAAGTACCAAAAATTAGGAGAATAGTGGCCTCCAGGGCAGAAGAGGGAGGATGTAATTGGGAAGGAGCTGGTGAAGTGCTGGTCACGTTCCATTTTATGGCATGGGTAGTTGTCACACAAGCTGCTATGGAGTGAATTTTGTCCCCTCAAAATTCAAATGTTGAAACAAACCCCCCATGTTGCTATGTTGGAGATTGGGCCTCTAAGGAGGTGATTGGGTTAAATGAAGTCACAAGAGTAGAGCCCTTAAGTGGTAGAACTGGTGTCCTTATAAGAGAAAGAGACAGCTTTCTCCCCTTTGTGTGAGAACACAAGGACAAGGTAGACATCTATAAGCTAGAAAGAGAGTCTTTCCCAGACCCAACTATGCTGACATCCCAATCTCAGGCTTCAACCTCCAGATTTGTGAGAAAATAAATTTCTGTTGCATAAGCCACCCAGTCTATGGTATTTTGTTAAGGCAACAAAGGCTGACTAATACACAAACATTTGTTTTAAAATAATTAATTTATGTTTTATGTACATTTTTATTATGCATGTTACATATCATAATAAAAACCCAATAATTTTTTAAAATAAATGACCATTGCAAAAAATGCAGAACACTGAAAATATAAATCAGAAGATGGTTACTAAGTAGAGAAATCACAAAATCATGAAATTATATTTAATATTGTTGAGATCATATAGCATGTAAAATGTCTATGCTTCTTTTGGAGTTTTAATGTAAAACATTTTTTTGCAAAATGAGTTATTTGGGAGTTATTTGGAGATTGTGGTTAAATATGGAAGATAGAATGCATATGTTTTACCCCACCCTCTCAAAATGCCAATAGAATGACAGCACGGGGGAAAGAACAGTATAAATATACAAGAACAAAGAGAAAGAGAATGGAAAACACAGAGGACAAATGATGTCAAAACACTTGGAAGACCAAAAGTGGGTGGAGAAGTGGTAAATCACTTAGCAGATCAGGGTTTGTTAAACCACGGTGCCTCCAGAGGGGGATAAAAATGAGAATAAAGGCATTTTACCGCCCCCGCTGCCCAACAAGCCCCAGAGGCCCTGCAAGTGTTGACATGATACAAATAGCAAGAAATGGAAGCAGAAAGAGGAAAGAACATGTGAGCAGAGTATGTTCACTTACTGCTGCTTCTATAATAACAAGAAGTTAAAGAGTATTATTTAAAGTTGACAAATAAAGAAATGGACAAAATAATTACACTTGACAGTACAAAGGTAAGCACAAAAAGATATTATTAAATACAGTCAGATAATGGATAAAACAGAGAGGAAAGGAATCACCAAATGGAAATGAGAGATTTTGATTAAAGAAATAGTACACTAAGGACAATGTATTACACGGTTTTAATTATAATTATAAATTTATAATAAGTTTTTACTTATAAAGACAACCACTAGGATAAAGATACAAACTCTTTAGTAATCTATATATTTGTAACAAATAATATGACAAACTCTAAATACTGGAAGAATTGTACATCAAAAAATAAAAAGCAAATAAGAGATGCCAAATAATGAAAAGGTACAAAAGTAATTGGGGTTTCAGACCATAAATATTAAATCATTATAACTAGGCTCAAACACATCTTTATTAATCAAAGTAGGAACCATTACAATCAACACATTTTTGCCAATGAGAAATGAGTTTGTTTATTCCTGTAGCATAAAAATCCATGCTTTGGGATTCAATGAACTCTTGGAAGGCATTTTCTGCATCCCGCTGGTTATGGAAGTGTTTCACCTGCAAAAAGTTGTTCAGATGCTTGAAGAAGTGGTAGTTGGTTGGTGAGAGGTCAGGTGAATATGGTGGATGAGGCAAAACTTCATAGCCCAATTCATTCAACTTTTGAAGTAACTGTTGGTTGTGTGACATGCAGTCGGTTGTTGTGGAGAAGAATTGGGCCCTTTCTGTTGACCAATGCCAGCTGCAGGCCTTGCAGTTTTCTGTGCATCTCATCGATTTGCTGAACATATTTCTCAGATGTAATGGCTTTGCTGGGATTCAGAAAGCTGTAGTAGATCAGACCGGCAGCAGACCACCAAGCAGTGACCACGACTTTTTTTGGTGCAAGTTGGGTTTTGGGAAGTGCTTTGGAGCTTCTTCTCAGTCCAGCCACCAAGCTGGTCATTGCCAGTTGTCAAGTAAAATTCACTTTTTATCGCACATCACAATCTGATTAAGAAATAGTTCATTTTTGTTGCATAGAATAAGAGACGACAACACTTCAAAATGACGATTTTTTAAACATTTTTCCTTAGCTTATGAGGCACCCATTTATGGAGCTGTTTCACCTTTCCAATTTGCTTCAACTGCCGAATGACCGTAGAATGGTCAACGATGAGTTCTTCCTCAGCTTCTTGTGCAGTTGTAAGAGGATCAACTTCGATGATTGCTCTCAATTGGTCATTGTCAACTTCCAATGGCCAGCCACTACTCTCCTCATCTTCAAGGTTCTCATCTCCTTTGCAAAACTTCTTGAACCACCACTGCACTGTACCTTTGTTAGCAATCCCTGGGCCAAATGCGTTGTTGATGTTGAGTTGTCTCTGCTGCTTTACAACCCATTTTGAACTCAAATAAGAAAATCGCTCGAATTTGCTTTTTGTATAACATAATTTCCATAGTCTAAAATAAATATAAAATAAACAGCAAGTAATAAGTCATTAGCAAAAAAAAAAGTGAGAAATGCACATTAAAATGATGTATAACATGGTCACATTTATTTAAGAATGTATTCCAATATCAAACGGCAAATTTCAACAATGCAAAAACCACAATTACTTTTGTACCAACCTAATATTTTAAAAACTAAATACGTCATATGAAATAATATAAACAAGCTAAGACTAAGCAAATCTGTCATGTCACTAAATATAAATGGATTTTACTCACCTATTTTTAAAAATGCCTATTACATTACAAAGCAATACCCAACTCTATGTTGAATAAAAAAAATACTCCTCACGGTGATACAGAAAGGCTAAAATTATAGAATTCAAAAGACATAAGCAAAGGCAAACAAAGAAAGCAGGATCCATGATTTTAATATCAGAGAAAGTGGAATCTCGGTCAAAAAGCATGAAATAGGACAAAAATTGCACAGACATATTAATATTAAAGACGATAGTAATCTATATCTTTGCAACAAATAAGATGGCAACAATATTTATAAATCAGAAATTATTAGAGTAGGAGGATAAATAAAAACATTGTAGGAATAGAAATTTTCAATTAATTTTAGCCAATAACAGATCAAATGTATTAAAAAGTATGTAAAATTATAGATGACCTAGTAATATAATTAATATAATGTATCTGATTAATATATAGCAAACTACCTTCCCTAACAATAGAAAATGCACCTCCTTTTCAAAAAACCAAACATATCAAACCAAATATTAAGCCATGACAAAAACCTCAATAAGCTCCAGAAAGTGGAAATAATATAGAAAACATTCTCTGATCATAATGAAATAGAGTTAAAAAAAATAACCAAATTTAATGAAATGAGAGGACTTTCCACTTGATAATTTTTAAAAATCTCTCTTAAAGAATTCTTGGACAAAAGACAAACTATAAACTAAAATTATATAATTTCGAGGGAAGGAAAGATAAAAAACAGTTTAATCAGAACCTAGAGAGTGCAGTTAAAGACATACATATTCAGGGAATAAGTCACAGCATGGAAGCATAGAATATTTATATCAGTAAAAGCTGAAAAGAAATTATTTAAAGACCTAACAAAACTAGAAAAAGGATAACAAATGAAAGAAAGAAGTTAATAATTATAAAAAGCAATAAATTACAAAATAGAAAAACAGTAGATCTAATAAATCCAAATGCTGTTGTAAAAATATGCACAAGACCTTTATACTAAAAAATAATACAGCATTATTGAGAGAAATTTTAAAGTACCTAAATGAATTAAAGGATGTCTCATGTGACTGGATTAGAAGATTCATAATTGTTAGAATGTCAGTGCTGCCCAAATTAGTCTATAAATTCAATGACATCCCCAAAAAAATCCCAGCAGCAGCTGTTTTATAGAAATTAGCTGAACAGATTTCATTTGCATGGATGAAGTGAAGTTTATTTAACTATTTTCCTATTGATAGACATTCACATTTATCATAATTAACATAATCCTCATATTTTGCCACCACATATTAATTTTTTGGTGAGTATCCTTACACATGTATCTTTGTACACATAATATGAGTAAAATTAAGGATGAATTTCTAGAAGTAGAATTGCCAGGTCAAAGGGGTGTACATTTTAATTTTTGAGATATTGCCAAATCGTCTTCCAAAGTTGGATCCTTTCCATTCCCAGTAATTATCAAATCCATTCTTTAGGAGATTTTGTATTAACTGAACTGTTGACATAATTCCCTTTATATGCACACTATGAAAAGTATAACATATACAAATCTACCTATATGGTCACAATCCTGGGAATTTATGTGACAGGTATTGGATGATGTTCAGCATCCTTGGCCTCGTGGAAATAATCAGACTAGATTTTCTTTTTGAAATGCAAATTGATAATAGCCATTAGTTGTTTCCTATGTTGCTGAGAACCCAGAGGAAGATAATAAATGTCCTTGGGATAGACTGGGGCTCCAAAGATCATGCTGTATACTTTTGAAAAAGCATTAAATTGTAGCCAGTGAGGAATCAGAAACCAATCTTATTGCCATGTTCTTAACACAATCTTTTTCCTTATCTTCCCTCCCTTCCATTATTCCTTGCTGAAACATTAGCTTTGCACAATTACCTGCTGATAATGGTAGCTCTATGTCTTTCTGCGTGCCTGCCTATCTGTCTGCTTGTGGAAAGATAAAGATAGATATTCCATTTTCTAATCCCCTCTTTTGCTTGGGAAGAAACTAGAGGTTCTTCCTCAGATTGGGCACCAGAAGGTAAAGAGAGCAGGGGGTCAAAAATTGTTAAGAAGGGCAAGACTAGAACTTTCCTACTCTCCCCATGCCCTGGTTTCAGAGAATCATTGAAGGGGCTCTTTGATGACCCTAGGACACTCACCTCTAAGCATAACAATATTCTGGAAAACCTTTGACAAATTATAGCAATTTGGTTGTAGAGAAGATAAATGTTTTCTCACCTGGCAAAGAACATGTGAAATGAAGCTGAATCTACTGCAAACGGGGAAGTGGGTGTTTAACACTGCATGCATAAAGTATTGATTTGTATCTTTTCAAATTTATGGAACTGTTATCATTTTATATGTGTGCTTTATCTGATGTTTTAAAATTATGTTTTTGGGATTTGCCCAAATGGATACATGTAGACCTAATTCATTCATTTTTTATTGCCATATAGTATTCCATAAAGATATTATCCTGTGTTTTCTCCTCAAAGTTACAGTTTTCCTTTCTACATTTAGGTATTTCAATCTCCTAGAATTTATTTTTGTATGTGTGTGGTATGAGATAGGCTTCCAATATTTTTCCATATGGATTATTCCAACATTTTTTTCAATACATCATTTCCCAAGTGGTTCCTACCACTGCTTCTGCCATGCACCAGATTCCCATGTCTACTGAATCTGTTTCTCCCAGTAATCTCATTGTCACATCCTGAGTCAACACCACGTGGATTTAAATGCTCTATAGTAAGATTTGACCTGGTCGATTCACATGCCCCTCCTTATTTGTCTTCTTGAAAATTATTTTGGTTCTTGTTTCTTTACTTTTCCATATCAGTTTAAGGGTCACATTTTCCAGCTCTTTTCTTACAACTCTGTGGAAACTTTGTTTGGTAATGCACTGAATTGATTTATCACCACAGGAAGAATCGCCACATTTACAATACGAAGCCTTCCCACACATCAGTATGGCAGATCTCTTAATTTATACAGGTCTTCTTTAATATTTTTCTACATTTTATAATGTCTTTTTCCTAGGTACATTGCAATTTTGGTTGCCCTATTGAATACTTATTATTGCTAAACTGTCTTAATATTATTAATTTCTATATAGTGATTTTGCATCCAGCAACCCTGCTGAGCTCCTTAAAGTTAAATTTTTCTGTAGATTCCCTTGGACTTCCTATTTAGACAGTTATATTATCTGCAAATAATGACAAACTTGTTTCTTCCTTTATGATTCTTATACCACTTTTTTATTTTTCCTGTCTTATTGAATTGGCCGGGACCTTCAACAAGATGGTAATTCAAAGCTATGACAGTGAGCATATTTATTTTGCTCTTGATTTTAAAGTGTGTGCTTCTAATGTTTCACTATTATGATGTTTATGAGAGTCTCATAATTACTTTTTATATCAATTTTTTCCATTTCTTGTTTGTTGAGACTTTCATCATGAATGGGTGTTGGCTTCTATCAGTATTTTTATATTTATTGAGGTTATCATATAGTCTTTCATCTGTTAGTATAGTAAATCTGTAAACTAACAGGTTTGTTAGTCTATTTTTAATGGTTCCAGCTGATCATTTTGCTGGATTCAATTAGGTGTATTTCATTTTATCTATTTTGCTAAATGAGGCTGGCCTGTAATCTTCTTTCATAAACTGTCCCTTTGGGATGTTCCAACTCTCTCCTCAATTGTCCCTGGGAACTCCAACTTATTCTTACTTTGTTTAGCAGAGCAATTCATCTCTCTCCATCTCCTACAATGTTTCTCAACAAGTGATCTAAGGATCTCTATGCACATATAATTTTAAAATCTAACATGTAAGATTAATTATTATATTCATAGAATTTCTAACTTTTTATGTTTTCAAAAACAGTGTATGTGCTTCAAAAACTGATTTCATTCATAATGGTTAACATGGAGTTAATCATAGGCAGTTTATCTTGACTAATGTATTTTATTGTCACCTGACAGTTATTTACACTTGTCAGACTTGCATTTGCCATTATTACTGTCGCTTTGCATAATATCACAATTCAAAAAAAAGAGTCATCACTTCGAAAGTGGTTTTCTGAAGCATAACAATAATGATAAAAATAGAAATATAAATCCTACAATGTGAAATAAGGTCACAATGTAAGTAATGAATGGCCTCCATTGTAAAAATACAAAGCATGAATCTGTTTGTCATGAGACAAGTGGATAATGGATGTGGATAATCTCCCAAAACCAGACAAAAATATATAAGAAAATGGGAGCCATGACAATGAATAATGATAATTGAATTTTATTGGACCAATGATCCTTTTGTCCTAGTACCCAATTTATTATCTGCTAACGTTTTTGTCAAGTAATGATATAAAGCTATCAAAACTTCCATATTATTTTCCAATAAAGTTTATCAACCCCACTTTTAAACCAATTGAATATCTCTACTACAAGCACAAAATAAGGCTTTCCCAGTATAAAATTGTGAAAATTTTATGTTAAAGGTTGAGAAGACAACAAAACTATAGAGTCATCATTCATCCTCATATCAAAAATAAGCACAATTGCACTCCTGCTGGTAAGATACAAAACCAGTTTCCAAATCAATTACAAATATTGTGGTTGGAGAAGAAACAAGAAAAACAATTGGTATAATTCCTTAATTAGAAGTATTGCATTCCTTATAATGTGACTGGCATTTAAAGTCAATGACTGTTAGGTATACGAGCAGACATTTTATTTTATAGTTGAACAAAACTATGAATAAGTAAGTTGTCAGCATATCTTCACTATATATATGGGAAAGACTCAGTGACTTTTTGTTCTATTTATCATGAAGCTAATTTTAAAAGAAGAGCTCTCTGGTTTAATTAAAAATTAGTTTGAACCTATGAAACAGACTTTAAAAGGTATGTTGGCTTATGCACTAATGTTTGTAGCAGCGTTATTCACAATAGCCAAATATGTCCATCAACAGATGAATGGATAAAAAATGTAGTATATATGTAAACAATTGAATATCATTCAGTTTTAAAAATGAATGAGATTCTGATACATGCCACAACATTGACAAAGCATTGTGCTAAGTAAAATAACTCAAACGCAAAAGAACAAATGTTGCATGATTCCACCTTTAGTAGGTACCTAGAACAGGCAAATTCATGGAGACAGAAAGTAGAATGGAAGTTGCCAGAGAGGGGGAATGGGGAGTTATTATTTAATGGGTACATTTCTGTTCGGGATTATAAAAAAGTTTGGAAACAGATAGTGGTGATAGTTCCACAATACTGAATGTACTTAATGCCACTAAATTGTGTACTTAAAAATGGTGAAAGTGGTAAATTTTATGTTATGTATATTATGCATATTTTACCAAAACTTTTTTTTAAAAGAGGAAAGGTGTTAAATTGCCATTGAAGAAACTCCAGCCACATGGTCAGCAAGGAAAGGTATCACTATCCAGATAGAAGATGCTGCTCCCAGATCCCAATGCTAGTGCATACCTTATTCACGGAACGCAGCTGGGGGTCTGTAATATGCCTCCAGATTCTGATTTGCTGTTGAGGTACACAGTGGAAATTGTGAATACAAACTGATTGTGGTCACTTAGTGTACATCTCTTTGGCCTACTGGGCTGAAATGAAAAGTGAGTAAGAGAGGCTGCTCTCCCACACTGAATTGCTTTGGCTGGCAAATGGGAAGGGCTTACACAGAGTTTTAAAAATAAGGAATGAAATGTTTTAAATTACCATTTTTATGAATACAAGTTGGGTATCTCAGTGATCCATTCAGAATTTGAATATTATGAATCTTTGTTTTCAGAGCCTGAATATCGGGATTTGTAACAATCAGACATCAGTGGTGCAAGTGGCTCAACGGCTGAGAACTTGACTTTTTCCCAATACCCAATGATTTTCTTCATTCACCAGAAAAAGCACTTAGGAAATAACACATTGGGCTTATTAAAAATTACATCTGAATGCTGCAACCCAACATCAAGAAATACCTTGCAGAGCCAAATACCACCAAAGAGAGGATGCAACTCCATTAGCTGCTGTTGAGCTGGGGCACTTGCCTTCTGGTGAAGCACTGAAAATAGTCTTTAGAGAGCACTCTCTCCATAATTTCTGGGTCTGTGTTTGATTTGACAAACCAGAAATATCCATCAAAGCTTCCCAACAACCTATAATTCTGAGTTAAGATTTTCTAATTTAGAAAACATAAAGAGTGTGGGGGGGAAACCAACAAGATATGGAAGCTGACAGCTCAAGCAGCTATGCAACCAGATATAGATTCCTAAATGATTTCAAACACGATTTCTCCTCTCACAGATGTGAAAAAAAATTATTCACTTTTTAACTTGACTTTTACTTTTATTTAAGACATATATGTTCATAATTTAAAAGGTCACAGAGTTTTATAAAGATTCTGACAAAAAAAACAGCAATTTCATTTTTCTCATCTCTTCTTTCCAGAGGCCACCACTTTTAGCTTTTTTTACCTGTGTCTTCTGATACCTCCCTATCTCCAATAATATACTCATAATACTAATTTTTAATTTTTCAATTTCTGATGTTATTTATTGGCTTCCTATTATGAGAAAAAACTTAAGGCCACATCCACAAACTCATTTCCTCTCACCCTCATCTTCCTGTTACAGTTATATTGTAATCTTCAGTTAGATCATCAGATAATATATCCATTGTATGACTACATATATAATTACATTACATTGCTAAGTAAACACTGTTTACAGCTGTGTCATGCAGTGTACTTTGACTATATTTCCTTTTTGCACAATGTTTTTGCTTTCCTTGGAGTTAATAAGGATTTTATTTTTTTGTTTGCTCAGTTTTCTATTTATCCCTAATTTGTTCCCAAACTCTTCTCCAGATGTTTAATTGTTGCTTCAATTTATTTGCACACATCAGGTGCATGTGTATCATTTTTTTCCTATGGGAATCACAGCTAGAGTACTTCATCTTCTGCCTCATTCTGACCTAGATGTTTTCTAGGCCTGATGCACAGCTGTTTTCTAGAGATTTTTTCTTCTTCATTCTTTTTTTGGATTCCTTTTGCCTCTTCCCTGGATCGCATATATGTTTCTTGTTTGAATGGAGCATATCCTCTAGTAACATTTTTTTTTAAGACAAAGTCTCTCTATGCTGCCCAGGCTGTTGGCCTCAAACTCCTGGGTTCAAGTGATCCTCCTGCCTCAACCTCCCAAGTAGTTAGGACTACAGGTACATACCACCATGCCCAGCTTCTAGTAACTTTATTAGAAGGGTAACTAAGAAGTACATTTCCCAAGACTTCATATATTCTACCTTCTTTGACAGTTTGGCTAGGGAAAGACTTCTGTATTAGAAATTATTTCCCTCTTAATTTTGAAGGCCTTGCTCATTGTCTTCTAATTTCTTTCTAGACCTCCTATTAGTCAAACAATATACCTCCTAGTCTGATTCTCTAATTTTCAAACTTTTTATCACTTCCTATCCATTTCTGCTTTTTTGTTCCACTTCCTGTGAGATTTCCAAAACTTTATTTACAAGTCCGCCTATTAGTGTTTTACTTTTGCTATCATATTTTTGTTTTCAAAATTCCATTTTGTCTTTGAAGGTACTTTTTACATCCTTCTGCTCTTGTTCTATACGTGGAGCCTCTTCTCACCTCTTAACTCTCTAAAAATATTAGTACAGGTTTTTTTTTTTTTGTTTTTGTTTTTGTTGTTGTTGTTGTTGCTGTTGTTGTTGTTGTTTTGGAGACGAAGTCTTGCTCTGTCACCCAGACTGGAGTGCAGTGACGCGATCTAGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCCGCCTCAGCCTCCGGAGTAGCTGGAACTACAGGTGCCCGCCAAGGCGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGCGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTAATCTGCCCGCCTCGGCCTCCCAAAGTGCTGCGATTACAGGCGTGAGCCACAGCGCCTGGCCAATACAGTTTTATTTCTCTAACTCTTCTTCTGCCCTCTTTATTGTCTCTTTTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTCAGTTGTTTGGTTGCTTTGGTTTCTGGTGTTAGAAGCATCCTTCAAATTCCTGATGCTCCTTGGCTGGCTGTTCATGTTAAAAGTGCAGCATCTTCATGGGCTCTAAACCAGATTATCTTCTGGAAACTTAACGAGGAGCAGAAGTGAATAGGGAGGGGAGTTGTTTGCCTTTCAGTAGGTAAACTTTTCTTTATATGCATTTTTCAGTATGCAACTGTTTCCCTCTTCTTCAACTCTTAAGAAAATGAGCATCCAATCCATTTCTGGAGCAGAAACAGTTTCTCATTGAATGGATGGCATAAAGCAGCATCTAGAAAGCTCAACTGGTTCCTGTAAAAAACTTTCAAGCAATTCTTTCAGCCCTACTTATCACCCTCCCCCAAACCTTGCTTTCAGAGATACTTAGCATCTCTAAGTTCCAAGCAGTTCTGGGGCTCTGCAGCATAGCTTGGCTTGCTTCTGGGTATTCCCTGCTTCAGCCAGTTTTCAGCTTTCTTAGGTCTGCTCTGTCAGTTACCACTCATCAGTCTGCACTCCAGTTTCTAAAAACTGGGAGTTTTTAGATGAGCCTCCAGCTTCTTGCAGGATAGGGAGGCCCTGGCAGACATAAACAGGAGAATCCTTGCTCTTCACCCGCTACTGCCCTCTGCCTAACCCCAGAACAGCAGAAGCTGCTGGCTGTCCCAGCCACACACATCCCTCCCCACTCCATCTATTTGTGTGGCCCTTCTTTTTCCCTCAGTCTGTGTCTTGTTTCCTCTCCTTACCTTCTTCCCCCTCATCACCTTGGTGATATTCTTTGCATGTTTTTTCTTGGAGCAGTCAGAGCAGGTCATCCCTCCCTCCTGGAAGAAGGAACCCCCTGAGTACACTCCATGGCACAGTTGACACTCCTTTTCTACCCTCCTTCCCAGATATGTCTTCACTGTCATTTACACCTTTGAAGCCTTGATAAAGATACTGGCAAGAGGATTTTGTCTAAATGAGTTCACGTACCTGAGAGATCCTTGGAACTGGCTGGATTTTAGCGTCATTACCCTGGCGTGAGTATATCTCTATGCAGATCTGCATGTGTGATAGGAGAGGGGACAAAATGGTGGGTAGGAGACAGGGCTAACATGCAGGTCCCACTTGGACAGAGTGTGGAGACTCACACCATGAACGTTTGCTCCAAGAACCACTGCAGGAATATACCAGGAGAACTGAAAGAATTCATAGATCCTTTTAAATAAGTGGCACACTGCTGGAAATTCTGCAAGACAGGTGAAAAACTGTGAGCTCCCAAAGTGTGAGGGGGGGAAACCTGCCTCCAAACATACATCCCAACTGGGTAATCTGAAAATCCAGATCACAGGAGAAGAATTTACCCTTACCTGGAATGTAAATAAATTTAGGAAGCCACACAAAATATAAAAGTAGAAACAGCAAAGGGAAGTGCCTTGTAGGCACTCCCAGTCTCCAGCTTGAGACCAGGGAATCCATCCCTGACTATGTCTCACAGGCACCCTTGGGGAAGGCAGCCAGTGGAATCAGGGAGGGGTCACAGGATGAAAGAAGTTTCAACTAAAATTAGTAATAATTTCAACTGGGCACAAATTTTCTTGAGCAGAATCCAGGGGGCAAATGGGAACTGCAAGCAGACAGTGAGGGATGCGGTCCAAAAGTCATGCTTTCTTTCTCAGTGGGGAAGCTCACAGCCTGGGGCAAGGTCTGAGTAGGGCACTGCAGGAGCAAGACCAGCCTCACCAACTACATGAGAGCTAGGTGAGGCCTCTCACTAGCAGCTATCCCCCACTTCCCTGGAGAACTCTATGGCACAGCAGAGGCAGCCAAAATTCCCTCTGGAACACAACCCCACTGGCCTGAGAACCAACCCCCATCCACCACAGTGGCCACAGCAATCCCTACCCAAGAAGGGTCTGAGTCTAGACTCACCTACCCCTGCCCCCACCTGATGGTATTTCCCTACCTGCCTTGGTAGCTGAACACAAAACCCAGAAACTCTCGGAAGCTTTATGGCCCTGCTCATCACCTAAGAAACTAAAATATTTACCCTGGCCAACTTAGGGTGAGCTTAGATCCCCCTTCTACTAATGCACTAGTGCTCTCTTGAAAGCATCACCTACCGGCTGGAGGTCAAACAGCTCAGGCCATTAAAGCAACTCATGACAGAATAACTTTGATCCCAGGAAAAAGAAAACAACAACCAGTTGCACTGCCTGCAACATCCTGGCTAACCAGACGTCCTGAGTCTGTCCATATGACAACTTCACTGCTAACATAACTAGCATTCAAGAAAGCCAGCACACTAAATCTATCTACAGCCTAGGACTCTCACAGACTCTACTTCATTCCTCTGCCACCTTCACCAGAACAGATGCTGATATCCATGGCTGGGAGACTTCAAGATCAATCACATCACAGGACTCTTGCAGGCATTTCCCAGTACTACCCCAGAGCCTGGTAGCCCCTCTGTGTGGCTAGGCCCAGAAGAACAATAACGATCACTGCAGTCCAGCTCTCAGGAAGCCCCATCCCCAGGGGATTGGGGAGAGCACCACATCAAGAGATCACCCATGAAACAAAAGAATCTGCACAACAGCCATTCAGTTCCAGATCTTTTCTCTGAAATAGTCTACTCAAATGAGAAGGAACCAGAAAAGCAATTCTGGTAATATGACAAAAGAGGGTTCAATAACACCCCTCAAAAGATCAAACTAGCTCCCCAACAATGGATCCAAACTAAGAAGAAATCTCTGAATTGCCAGATAAAGAATTCAGGGCTGGGCACAGTAGCTCACGCCTGTAATCTCAGCACTTTGGGAGGCCAAGGTGGGCAGATCACCTGAGGTCAGAAGTTCAAGACCAGCCTGAAGCCAACATGGAGAAACCCCGTCTCTACTAAAAAATACAAAATTAGCTAGATGTGGTGGTGCATGCCTGTAATCCCAGCTACTCAGGAGACTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCAGAGGTTGCGGTGAGCTGAGATCGCGCCATTGCACTCCAGCCTGGGCAACAAGAGTGAAAATCCATCTCAAAAAAAAAAAAAAAAGAATTCAGAAGGCTGATTATTAAGTGATTCAAGGAGACACCAGAGAAAGATAAAAACCAACTTAAAGAAATTTTTAAAACAACAAGATATGGATGGAAAATTCTCCAGAGAAATAGATATAAAGAAAAAACAATCACAACTTCTGGAAATGGAAGACTCACTTAGAGAAATACAAAATGCAGTGGAAAGTTTCAACAATAGACTAGAACAAGTAGAAGAAAGAACTTCAGAGCTCGAAGACAAAGCTTTTAATAGACCCAAACTAAGACACACAAAAAGAAAGAATTTTTAAAAAAATGACAAATCGGCCAATAAATTTGGAATCATGTGAAACAACCAAACCTAAGAATAATGGGTGTTCCCAAGGAAGAATAGAAATCTAAAAGTTTGGAAAACTTATTTGAGGGAATAATTGAGGAAAACTTCCCTGGCCTTGTTAGAAATATAGATATCCAAATACAAGAAGCTCAAAGAATACCTCTGAAATGTGTCACAAAAAAGATCATCACCTAGGCAAATAGACATCAGGTTATCTAAAGTCAAGACAAAGAAAAGAATCTTAAGAGCTGTGAGACAAAAGCCTCAGATAACCTATAAAGGAAAGGTTATCTGATTTACATAAGATTAACAGCAGATTTCTCAACTGAAACCTTGCAAGCCAGAAGGGATTAGGGTCCCATCTTTAGCCTCCTGAAACAAAATAATTGCCAGCCAAGAATATTGTATGAAGAGAAACTAAGCTTCATAAGTGAAGACAAGATAAAGTTTTTTTCAGACAAACAAATGGTGAGAGAATTTTCCACTACCAAGCCAGCACTACAAGAAATGCTAAAAGGAATTCTAAATCTTGAAATAAAACCTTAAAATACACCAAAATAGAACCTCCTTAAAGCATAAATATCAAAGGACCTATAAAACAATAACACAATAAAAAAAGGAATTAAGGCAATAACTAGCATAATGAATAGAAGAGTACCTCACATCTCAATACTGATGTTGAGTGTAAATGCCCTAAATGATCCACTTAAAATATACAGAAGGGCAGAAAGGAAAAAAAAAATCCACCAACCAAGTATCTACTGTCTTCAAGAGACTCACCTAACACATAAGGAGCCACATAAACTTAAGGTAAAGAGATGGGAAAAGATATTCCATGCAAATGGAAACCAAAAGCAAGCAGGAGTAGCTATTCTTATATCAGAGAAAACAGACCTTAAAGCAACAACAGTTAAAAAAAGACAAAGAGGGACATATATAATGATAAAATAATTGGTCTAATGGAAAAATATCATAATCCTAAATACATATGCACCTAACACTGGAGCTCCCAAATTTATAAAACAATTACTACTAGACCTAAGAAATGAAATAGACAGCAATATAATAATAGTGGGGGATCAATATTCCACTGACAGCACTAGACAGGTCATCAAGACAGAAAGTCAACAAAGAAACAATGAACTTAAACTGTAACCTAGAACAAATGGACTTAACAGATATTTAAAAAACATTCTACCAAACAACTGCAGAATATACATTCTTTTCATCAGCACATGGAATATTCTCTAAGATAGACCACGTGATAGATAACATAACAAATCTCAATAAATTTAAGAAAATAGAAATTATGTCAGGTATCCTCTCAGAGCACAGTCGAATAAACCTCGGAGTTAACTCCAAAAGAAACCCTCAAAACTATACAAATACATGAAAACTAAACAATCTGCTCTTGAATGATCTTTGGGTCAACAATGAAATCAAGATGGAAGTTTAAAAATTTTTTGAACTGAATGATAATAGTCACACAACTTATCAAAACCTCTGGGATACAGCTAAAGCAGTGCTAAGAGGAAAGCTCATAGCATTAAATACCTACATCAAAAAGTATGAAAGAGGACAAATAGATAATCTAAGGTCACACCTCAAGGAATTAGAGAAACAAGAACAAAACAGACCCAAACCAAGCAGAAGAAAAGGAATAACAAAGATCAGGGCAGTACTAAATGAAATTCAAATAAAAATAATAATAATACAAAAGATAAGTGAAACAAAAAGCGGGTTCTTTAAAAAGATATAGAAAATTGATTGACCATTGGCAAGATTAACCAAGAAAAGAAGAGAGAAGATCCAAATAAGCTCAATTAGAAATAAAATGGGAGATATTACAACTGATACCACAGAAATACAAAAAAAAAATCATTCAAGGCTACTATGAACACCTTTATTTGCACAAACTAGAAAATTTAGAGGAGATGAATAAATTCCTGGAAATATACAACTGTCTTAGATTAAATCAGAAAGTAATAGAAACTCTAAACAGACCAATAACAAGTAGCGAGATTGAAACAGTAATTCAAAAATTGCCAAGGAAAAAAAGTCCAGGAGCAGACTGGATTCACAGCTGAATTCTATCAGACATTCAAAGAAGAATTGGTACCAATTTTATTGAAACTATTCCAAAAGATGCAGAAAGAGGGAATCCTCCTAAATCATTTTATGAAGCCAGTATCACCCTAATACCAAAACCAGAAAAGGACTTTTAAAAAAGAACTACAGACAATATCCCTGATGAACATAGATGCAAAAATCCTCAACAAAATGCTAGCTAATTGAATCCAACACATATCCAAAAGATAACACACCATGATCAAGGGGGTTTCATACCAGGGAGGTAGGGATGATTTAACATACACAAGTCAATAAATGCTGCACGTCACATAAACAGAATTAAAAACAAAAATCATATGATCATCTCAATAGGTGCAGGAAAAGCATTTGACAAAATCCAGCATCCCATTATGATTAAAACCCTCAACCAAATTGGCATAGAAGGGACATACTCCAAGGTAATAAAAGCCATCTATGACAAATCCACAGCCAACATTATACCAAACAGGGAAAAGTTGAAAGCATTCCCCCTGAGAACTGAAACAAGACAAGGATGCCCACTTTCACCATTTCTATCCAACATAGTACTGGAAGTCCTAGCCAGAGCAATCAGACAAGAGAAATAAATAAAGGGAATCCAAATCAGTAAAGGGGAAGTCAAACTGTTGCTGTTCACCAATGATATGATCGTATGCCTAGGAAACCCTAAAGACTCATCCAAAAAGCTCCTAGATCTAATAAATGAATTCAGTAAAGTTTCAGGATACAAAAACAATGTATACAAATCAGCAGCACTACTGTACACCACCAGAAACCAAGCTGAGGAACAAATCAAGAACTCAATCCCTTTTACAACAGCTGCAAAAAAAAAACAAAAATAAAAACAAAAAAATCTTAGGAATATACCTAACCAAAAAGGTGAAAGATTTCTACAAGGAAAACTACAAAACACTGCTAAAATAAATCACAGATGACACAAACAAATGGGAACACATCCCATGCTCATGGATGGATAAAATCAATATTGTGAAAATGACCTTACTGCCAAAAGCAATATACAGATTCAATACAATTTCCATTGAAGTACCACATCATTCTTCACAGAAATAGAAATAACAACCCTAAAATTCATAGGGAACCAAAAAGGAATCCACATAGCCAAAGCAAGACTAAGCAAGAATAACAAATCTGGAGGCATCACCTTAGCTGATTTCAAACTATACTACAAGGCTATAGTATAGCCTGTACCAAAACAGCATGATACTGGTATAAAAATAAGCACATAGACCAATGGAAAAGAATAGAGAACTCAGAAATAAAGCCAAATACTTACAAGCAACTGATCTTTGAAAAAGCAAACAAAAACGTAAGTGGGGAAAGGACACCCTATTCAACAAATGGTGCTGGGATAATTAGCAAGCCACATGTGGAAGAAAGAAACTGAATCTTCATCTCTCACCTTATACAAAAATCAATTCAAGATGGATAAAAGGCTTAAATCTAAGACCTGAAATTATAAAAAATTCTAGAAGATAACATTGGAAAAACTCTTCTAGACATTGGCTTAGGCAAAACGTTCCTGACCAAGGACCCAAAGCAAATCCAACAAAAACAAAAATAAATAGATGGGATCTAATTAAACTAAAAAGCTGTTGCACAGCAAGAGAAATAATCATCCAAGTAAACAGACAACCCATAGGATGAGAGAAAAATCTTTGTTATCTATGCTTCCAGCAAAAGACTAATATCCAGCATCTATAAGAAACACAAATAAATCAGCAAGAAAACAAACAAATAATCCCATCCAAAAGTGGGCAAAGGACAAGAATACGCAATTCTCAAAAGAAGATATATAAATGGCCAATGAACAGGAAAAAAAAATGCTCAACATCATTAATTATCAGGGAAATGCAAATTAAAACCACAATGAGATACCACCTTACTCCTGCAAGAATGGCCATAACCAAAAAATAATAGATGTTGGCATGGATGTAATGAAAAGGAAACAATTTTACACTGCTGGTGGGAATGTAAACTAGTACAACCACTAAGGAAAACGTTATGGAGATTCCTTAAAGAACTAAAAGTAGAACTACCATTTGGTCCAGCAATCCCACTACTGGGTATCTACCCAGAGGAAAATAAGCCATTATTAAAAAGACACTTGCATATGCATGCTTATAGCAGCACAGTTTACAACTGCAAAAATATGGAACCAGCCTAAATGCCCATCAACCAACAAGTGCATAAAGAAAATATGGTGTATGCATACCATGGAATACTACTCAGTTGTAAAAAGGAATGAAATAATGGCATTGGCAGCAAGCTGGATGGAGTTGGAGACTATTATTCTAAATGAAGTAACTCAGGAATAGAAAGTCAAGCATCATGTGTTCTTAATTACAAGCAGGAGCTAAGCTGTGAGGATGCAAAGGCATAACAATGACATAATGGACTTTGGGGACTTGGGAGGAAAGGTGAGACGAAGGTAAGGGATAAGAGACTACACACTGGGTACAGTGTACACTGCTCGGGTGATGGTGCACCAAAATCTCAGAAATCAGCGTTAAAGAATTTTTCTGTGCAACCCAACACCACCTGTTTCCCAAAACTATTGAAATACAAATAAAGAACTTATTCATGTAACCAAAAACCACCTGTTCCCTCAAAACTATTGAAATAAAATAAAATAAAAAGATTTGCATGGGTGTCCATGTGGGGCTTATGGGTGTATCCAGGCTCACACTCTGAAGAAGGTTCTTCATCTCAGTTCAGCTTCCTCATTTGGAATTGTATGGCCTGGGGTTCTTGCCACAGAGTACCGCTTCGGGGGTTGCTTTCCAAGATTAGTCCCATGCCCATGAGGAGATGTCAGAGACCTCAAATCTGAGATAACAGTTAGCCTGTCACAGTGGATCAGAAAAGGCCCAGCCTTGTACCCCCCTGACATGGCGGTCTCCTTCTGAGAAATCAGTGTCTGAGGAGACTGGTTGGTCAAACTCCTGTGTTTGAAGATCCGTCCACATGCAGTGTTGCCTAGATCACAATGCATCTTCTCAAAGGGTCCACTCTGAAAGATGTTGCAATAATGAAATCAGTGTCAGTGTCAGTCTATAGATGGCAGTGTCACTAGATTCTAAGTGTCCATTCTGCATCCTTTCTAGGCTAATGATACCCCAGGTTATCCTTATTTATGCTGCATGCATCCCCAGGACCCTTGCTTGCAGAGATGCTGACTAACTTCTGGTCCTACAGATATGTTGGCACAGCAATAGATCTCCGTGGGATCTCAGGCCTGCGGACATTCAGAGTTCTTAGAGCATTAAAAACAGTTTCTGTGATCCCAGGTGAGCATTTATTCACAGAGCATATGTTGGTTTTCACAGCCTAACTCCCTTCTCTAATTTTCACAAGAACCCTGTAAGGTTCCAGGGCAAAGACTATTGTCCCCACTTACAAGACAAAGGAACTGAGGCCTGGAGTATAGGAAACATAAACCCAAGGCCTCTTCACTAGTAAGAGACAGTGTGACCTGGAAGCCTATTGTCCCAACTCCTAGGCCAGTGCTCTTCCCACATACCACATGGCCTCACAATCAATACGTAACAGGAGGGGCATGTCTTCACTTTCTGTTCTGGTTGTGGGATTCAGGCAAGACCCCTCAGTTGAATTTTTATTTATACCTTAAACTAAAGGATTAAACTTGGAAATGGGGAGGAAGGGAGAAAGCAGTCCCAGTATCTTCACTGTTTCCGTAGGTCTAAAATCCTGTCTTTAGACACCCAGAGGAAAAAAGAATTAGGCAATTCTTGATTACTGAGAATTCAGAAACAAAAGTAGTCCATCAAAGCTCATTGTGTGGGGCCTCTAAGATCTCCACTGTCACCAGTAGCCCAGCGACTACTCTTCAGCCCCCATGAGGTTGATGGTGAGGATGGGGAGGCCAGAGACAAGGCAAACTTTCTGAAATGGGACTTGATGCTTCCCCTCTGTGGCCTGATAGGTTCTCTCCTCTCACCCCAAGAATTCGTTCTCTTATCTGTAAACAGAAACACCCATTCTAGAGTGGAAGTCTTTTCTACCATTTCTTGTCTGGCTCTGCAGGGAAAGATAGGAAACTCTCTGCCAATTCCCACTGGACACACCTCTTGAAGAGAGCCCTGGTCCTGCCCCTTCATATCTCTCTCACTTACTCTCAACTGGGAAGACCCTGAGTCTTATTTGCAAGACCTGGTCTGTCCATACTGTCTCTTCTGCCTCATCTCTCACCTCTCCTCACATCTCACCATGCTCTGGAATCCACCTACGGTTCTGCAAATACTCCACACCATTGCTCACCTCCCAGCCTTCTACTGTGCTGCCCCTTCTTCTGGGATATCCTTCCTCCTCTCTTTACTCAACTCACACTCATTCCTCAGGGCTCTGCCAAGGTTTTACCTCCTCCCTGGTTTTCCCACCTTCCTCACACTCCACCCACCCCATTCCCACCTCTGGAACTTCAGTTAAGGGCTGCTCCCCTGTGTGCCCATCTGCTCAGTAATGCTACAGACTTTCCCCTCAATTGTCAGACAACGGCACTCGACATGCTGCTGCCTTAGCCATCACTCAGCTCCCAGTCCAGGGACTGACACATGTAGTGCTCAGTGGATTATTTGCTGACCAAAATGACTAACCAAAAATAAATCCTAGATTGCTTCCCTGGTCAGAGCTCTTTCTGCTACCTGCGAAGCTGCTCTCCCAAAGAAGTTGATTTTACTTACCAGAGCAACAAATCATCCCTCCTTAAACAAGCAGGTGTATCCTTCTTTGCTGCCAGCTGTGCTTGGCACAAGTTCTATTTATGCCCCAGTGACTGTCTCAGTGTTTCTTATAGTCTCTTTCTATTGCTAAACTCACCTCTCTGTTTCTCTGTGTCTCTTTCTTTTTCTTTCTCTTGGTCTATTTTTGTTTCACTCTATCTCTCTTGCATCTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACACACACACACACACACTCACACACAGTGAGGTTCATGTGTCCCAAGTCTCAAGCTGCCTCCCAGATGCATGCAGAAAGACAGATGCCAAATTACTGGGGTGAAGTTAGTAGCTTCATGCCTAGCACAGTGCCTGGAAAAAGTAGGTACTCAGCATAAATTATTTAAATTGAATTGCTAGTGTCAGGAATTTTATTAGCCTCAAGATGATAAAAAATGCATTGCCTGGGTCTCAGTTGCATCGTCTTTATAATGAGAATAGCCCCTGACTCTCTGCCTGCCTCTCAGAGTAAATGCAAGCTTCGGATGAGAAAGGTCTTGGAACTGCCCTGGAGTGTGTACATACTTCACTCCTGTATGTAGAGATGAAGGAGGATGGCTAAGATGAAGAAGTGTGCTCCGTGTGTGCTACCTCATCCATGTGGTCATACCACTGTCATCTTCCCGCAGGCCTGAAGGTCATTGTGGGGGCCCTGATTCACTCAGTGAAGAAACTGGCTGATGTGACCATCCTCACCATCTTCTGCCTAAGTGTTTTTGCCTTGGTGGGGCTGCAACTCTTCAAGGGCAACCTCAAAAATAAATGTGTCAAGAATGACATGGCTGTCAATGAGACAACCAACTACTCATCTCACAGAAAACGTGAGTGGAGTCTCTTCCATAGGGACCAAGGTCTGGTTGGACCCTTGGTATCATATAGGGACAGGGGATATGGAGCCCCTGTGTGTGTGTATAGACATATATTTTGACTCCCCTTTCCTGCCAAAATGGTTCTCTTGAGTTGTGTTTTACTTTTGGGTATTCTATTTCCTGTTGCTCAGCATCCCTCTTGCAAGGAAATGAGCCATAAATCTAGAGGCAAGTCATTCTACCTCTGCTGGGGGAGATTGAGAGCCGAAGTCACTGCTTAGCTCTTCTCTTTTCAGATGTGACTTTACAACTTCCATGTGGCCCAGCATGCCCACTGAGTACATGTAGCCAAAGGAGCTCACTTTGGAAGACTTGGGAATATTGCATTCACCACACAAGGCTTGGGTTGGAGGGTTCAGAACCATCTGTGAGGCTTTCTTTTCTTCCTTTCTTCAGCAGATATCTACATAAATAAGCGAGGCACTTCTGACCCCTTACTGTGTGGCAATGGATCTGACTCAGGGTGAGTTCCCAACTTATGACAAAGCACGAGTGCCCAACAATTCCTTCTCCATCTTGTCCTTGGCATATTATGGGCACAGATGGGGTTGCAGGTCCTGGGAAAGATGTATAAATAAATAAATCATGGCCTTTGCCAACCCAGAGCTCATGGGGAGGGAATGTATGAGTTAGCTTTCGCTGTGTAACAAATGACCCCAAAATTTTGTGAATTAAAACAACAATGTATTTAATTCATGATTCTGTGCATCAGCATTTTGGACTGGGATCAGATGGGCATTGGGTGACATTGACTAGGCTCATTCATGCATATGCAGACAGCATATTAACTTGGAGATGGCTGGTCTAGGACGTCCTGTCTCTGCTCATGTGGCCTCTCACCCCCAAACAGGTTAGCCTGGGCCTGTTCTCATGGCAGTGTTCCCAGAAAAACAGTGGAAGTTTGCAAGGCTTCTTCAGGCCTAGGCTTAGAACCAACACAATGTACCTTTTGTACATTCTGATACATTCTTGACTAAAGCAAGTTGCAGTCCAGTAGAGATCCAAGAGACAGAGAAATAGACTTCATCACTCAATGGAAAGAGCTGAAAAGTCACATTGGAAAGGGTGTGGATACATAGAGACAAGAATTATAGCCATTTTAATAAATAATCTACCCCAGGAAGGAACCAATACTTATTTGTGTCAAATGCTCTACACTTAATCATCACAGCTAACTCCATTCTATAGCCACATTACCCTCACAAGTAGAGGAAAAAACACATGTATCAGACTTTGCAATTTCGAATTCTTACAGAGGCCAGATTCATAACAACAAATGAGTGGAGTGGGCCAGGAGGGAACCTAGGAGCTGAGAGTTCATGCCTCCTCTAAAGGGGACAGCCCCACTGCTCGGTCCAGCTTTTGGTTGCCATGTGGAAATGTGGGTTCACTGTTTCCAGCTCTTCCAACTTTTAAAATAGAGATAGGAAATTTAGATTTTTATATAAAATCTCCCAACCGTAAACATTGGCTACCTATTCCAAAAAATGTCTACATATTGTTGGGCCAAACAAAATATTTCTGTGGGCCAAATCCAGCCTATAAGCTGCTAGTTTGCAATTTCTCATTCATAGACATAAAGATGCTACCCTGTGGAAAACAAGTTCTTTACTAGAGGCTTCATAGGCTGGGCAAACACAGTGAGGTGGCGGTGTGAGGCAAGAGAGAAAGTAGGGAAGGCTTCCTGGAGGTGGTGTTTGAGGTGAGCTTTCAAGGGCAGGTAGGATGTCTCCAGGCTGGGTGAATACGAGATAGTTTCAGCGAAGACCATTCCAGGTGGCAGAACAGAATGTGAGACAAAGCTAAGGGATGAGGACCCCTCAGCTCTGCCTGCTGCTTTGGCCACTGGAGGAAGGCATGAGCAGAAAGACTTTAGGCAAAAGATCAAAAAGGCCCATCTCACCCACCTCCATGGCTGAGAGCTTGGAAAAGCAGAGCTCACAACACCCCCAGTGCCTGAGAGGCTGACGGCTCCATGTTGCCCAGGTCGACCTGTGTGGAGCCTGCATGAGGCTCCACCTTCATCAGCTCACTAGGAAAACTATCTCAGTTGGAGCTGAGGACTGGAAACAAGCAGGAGCATTTGGTCAACATCAGGTTCCTTGGACTGTTCTTTCACAAATGCACTTGGCATTTGGTTCTTTGGCTCATGAGAGGGTTGTTCTGACATCAGTTTTCATTTGCCTTTGACTTAAATGTAAACCAAGAGCCCCTCAGCATCCTTCTGTCTAGATAATGGCATTTCTCAAGGCAATGGCTCCAGAGCAGCTCAAGTCCTTTTTGTGGTAAAGTTGTTGGGTCCATCTACACACCTCTACAGCTCAGCTCCCTGAGAGGCCAGGCCTGCTATTGGGCAGGTGCCAGCCAGGCAGTGGGGGAGTGGACCAGGCTGGGATGGCAGAGAGAGGCCAGCTCAGGATAACGGACAGAGTGAACCCTGCCGGGGCATGCCGTGAAAGCTAAACCTGGGTCCTCTGTGGGGCTGCAGAGAAGCCAGAGTCCTGAGGAAAGGCCCACATTGGCCAATGGCACTGTGGGAGGAAGGTCAGGGGTCTCAAGGCAGAAAATGGATACCAGAAAATAGTTGATAATTTTCAGTGTTCTTCCACATATATTATTCCAGTTTATCCTTTCAGCAGCTTCCAAGGTTAAAAACAATAGGAATTTTCCTTTGCAGTTTATATACAATATTTACTATGCAGAAGTGCAAGACACTATACAGTGATCTCATTTAATCCTCAGAGCAGTGAACTGTTTATATGCAGTGGCTATGGTGGATTTCTACAGAAGACATAGAAACAAGGCTGCCCTCAGAGTTAATTAAGAAACCTACAGGAAATGGGTTGCCTAGCCCCTTGGAAGAGCAAACCAAGAGCCTATCTTTAAATGTGGGTCTACAAAGCCAGGGAACTTGCAATCTTCAATCGAAGTGGAGATAGACTTGGAACTGATTAGAATTAAGTTGTCAGAGCTCTAATCCATGGTATGGGTTGTTCACATGGATGTCTGGGATAAGGAGAGCCCCAGAGGTTGGAGTTCATGGAGAAATCCTGGGAGTAAAAGAATTCCATGTATTCACAAGGAATGCAGGTAAATAAAACAGGAGAAAAAGAAAAAAATTTAAAAAAATAATTCTGGACTTCAAACGAGGCATTGCTGTTTCATTGATCAGGCCATCTCTGCTAGGGTGGATGAGAGCACTTTACGCTCCCTGAGCTGACAGATGCTTCTTGACATCGACTCATCCTCCTATTATGAGAGAGAGGAAGGAGTTCAGCATTCCCACCTCAATACTCACCAGGCAGTCATTTTTAATCTGGCCTTCCTTCATAGGCCATCTTCAGGAGGAGGCTAGGCTGACTCAAGTATTGGGCTACTTGACAGTTATATTTCTCCTTTTAATAAATATATTGACACCTGCGTAGTTTTAGGCAACTGACTTCTTTCTGACCCCCAGATTCCTTATCTGTAAAGAAAGAACATTGAGTGATTAAATTATAGTGTTTGTAAAGCACTTAAAGGGAGAGAGAAGAACACAGAAAGTGCTCAGTAAGTGGAAAGTGTTATTTCCACTGTTACATATAATTAATCAGTGAACTTGGCTAATGTGAGACCTCCCTTGATAGACAACTCTGCAGATGATGCCAACAGGCTCAGGTTTTAAGTCTGCGTTATTGCTACTGACTCTCATTTCCCTGGTATGAACTGGCAAAGAGCTTATAGTATCGTGTCTGGGCAGTCAGCTGCCTTCCCCAGGCCACTCCCCTGTGTATGAAATTCTGACGTGAAAGTATTAGCGTCCCCGTTTTATGGATAACAGAAACCAATACCTAAACAGACCAAGTGTCCAAGATCATCCTGCTAATAAGAGGAAGACTAGGTCTTGAACTCTGATCTCTGACATAAAACTCTGTGGTTGTCTCGCAGCCCTGGGCTACCTTGTCTGCAATAAGCAGGGGATGACCTTCTCTGTTCTTGCTGCAGCCACTGCCCTGATGGTTATATCTGCCTTAAAACTTCTGACAACCCGGATTTTAACTACACCAGCTTTGATTCCTTTGCTTGGGCTTTCCTCTCACTGTTCCGCCTCATGACACAGGATTCCTGGGAACGCCTCTACCAGCAGGTACTGAGCTGCATTCCCCCACGCAGACTTGGTTGGAGGCTGGTGGGTCAGCTGCATGGCTGTGCTTTTCCTGGAGGAGGCTGACTTAAATTCAGGGGTCCATGTGCTATGGGTACAGGTTTCTCTTCTTTCCCTTGAACCTGCAGACCCTGAGGACTTCTGGGAAAATCTATATGATCTTTTTTGTGCTCGTAATCTTCCTGGGATCTTTCTACCTGGTCAACTTGATCTTGGCTGTAGTCACCATGGCGTATGAGGAGCAGAACCAGGCAACCACTGATGAAATTGAAGCAAAGGAGAAGAAGTTCCAGGAGGCCCTCGAGATGCTCCGGAAGGAGCAGGAGGTTTGTGAGGAGTGGAAGCTTTGTGAAGAAGGTTGCAGACTGTCCATTCTTGGATACTGTTCTGGACTAGCCACTTCAATTAGGGCACCGTTTAGGGAGCTATAGGAGGAATCATCTTGCCTCAGGGACCATATTAGATCAGGTTTTCTCAAAGAAGAAGCTAAGTGAGGATTCTTGTGCAAGTGACCTATTGAGGGAGTGCTCTCAGGAGAAAGCGAATGAAGCAAAACAGAATGGGGCAGAGGGTGGAACTAAACAAGGAGTGTTCTCAGCTAGAGACCAGCTTCAGCTTGATCCCATGGGGAACTCTGGGGCATGACCTGTACCACAGTTAGTCCTACCTTGAGGCAAGGGGGCCAGCTTTTTTTTTTTACCCTCCCCATACCTATCCCAGAAGCAGGCAGCCAAAAGAGACTTTTTTTGTAGAATGCTGCAAGTATGAGCCATCAGCTGCTGCCATGCAAGCAGCTCGGGATAGCTGTGCTGACCTGGTGAAAGGGATCTGGGTGGGGCACCAACAGAGTACCCCATGAATGAGTGGGGGTGACCCGAGGTGTCTGTTCCTGCCATGGCCTAATCCCACCACCACCCTGGTCCTAACCCTTCAGCTTCTCTGTTTCAGATCTCTCACCCCTGGCACCCCATGTCTGGGGGACACATTCATGCTAAGTCCAAGCAAATACTATTGGCTACACCTGCTGTTCTCTCTTCGCAGGTGCTAGCAGCACTAGGGATTGACACAACCTCTCTCCACTCCCACAATGGATCACCTTTAACCTCCAAAAATGCCAGTGAGAGAAGGCATAGAATAAAGCCAAGAGTGTCAGAGGGCTCCACAGAAGACAACAAATCACCCCGCTCTGATCCTTACAACCAGCGCAGGATGGTAAGAGGCTCAGGTTTGTGCTCTAAAGATTACCCACCATTGCAGCTGCCGTAGACAATGAGTTGGAGTGGGAAGAGGTGTGTGGCAGAGAAAGAGCTGGAGGCATAGAGCCTAAAAGGATCCAGACACCCCTCCAAAATGGTGTCTTAAACTAGGAAAGTGTAATTATAGATGGGGAAAAGGGGTGAATTATAAGGACACCTTAGGGAAGTGGATCAACAAGATTGGGATTGTGGACTGTGGAAAAAGGAAAGGGGAGTCCAATTTGACTCCCAGGTTTCAGGATTGGGCAATCAAATGGGTGGTGATTCCAAGATGAGGACACTGGAGGAAGAACCCCGGTTTGTAGGAAAGGTGGGAAGCCCATACTGAACATGCCAATCTTAAGGTGTCTGCGGACATCAAAGGTCAGCTTCCTTCTGAGGCTCTGGTCTGCAGTTCTAGAGTGCAAGGTTCCCTGTCCGTGGTGCTGAGATCCTTCATGCACCTTTTTTTTTTCTGATGCGTCGAGGTGGTTTTTATTTTTTTACTTAGTTTTTTTTAATTGACACTTCACAATTTTATATAATTATTGGGTATAATTTCTGACCTCCTTCCTAAGGTCCTGCAATTCAAGCTGTCACCTGTTGTTATCAGAAAAGTTTCCAGCACCCATCTCTGTGACACAATCCAGCCTGGGAATGGCTTCAGCACCTCACCTCAGACCCTGTAGCAATTCTTTTCCCCCCATAATAACTTTCTGCCTCCCACTCTAAAATTTGCACTGCAACTTTCCTCGCCACGCTTCCTCCCACCACCTTCCTGCCCCTTGAAAGTCCTTTTACCCCAAATATGCCCTTTCTATAGGTCTGACAGATAATATCTGCTGTGTGGGAGGGTGGCTCCACACTAACAGTGTGGAAAAGGGGATCAACATGGCTATTTTTTTATGCTTTATACCTCTCACTTCCCTCTCAATCTCTTTTCCTGTGCTAAGCCGAAGGATATTTTACTTTTTTGCAATACCATCTCTCCAACCCTCAGATTTATGTGTGCATATACAAACACAGACACACTTGCATGCACACTGACCTCATGACACATCCACACTTCAGCATTTATCTGCATCTGTGCCCTCCTCTTTCTTGCTTTGCCCCTTTTGAGCAGCACCTGGCATACATGGGGCACAGTTCAGACAATCACAGAACAGGATGAATCACAGCTTCCATGGAAATGTCAATGACACTGAATTGGGCTTTAATTTCCCCCATTCTGTCAGCCCCACACAGATTAGCACTTGGATGTCCAATAATTGGTATGTGTGCTTGTCTCTCCAGTGATGCTTACTTTCAAAGGGAAAAAAGAGGAGCTGTAACTTGTTCATTCAACAATACTAGGTGCTTGGTATAAAGAGATGACTAGAACATGTTCTTTTCCCTAAGGATTTCCATTTGGGTGGAGGAGGCAGATATAAACACAGCTATGTGTTAAATAGTGTATTAGAGACACATACATGGTGTTTTGAGAATCAGGGAAGAAACATCCACTCTTATGGGGCTGGATAGGTATCAGAAAAGGCTTCCTGGAGGAAGGCATAATTGGGATCAACAATAAATAGCATGTAGGGGTTCTTCTAGTAATTGAAGACTTGAGAGAGTTTGGGGAGGAGTATTTTCCTGGCAGAAAGAACAACATGTATGTGCAAAGACAGGGAGGCACCATTTAAGTACTTCTTTAATGCTAATTGATTTATTGGAGCCCACTCAGGAAGGCATCCTCTCCCTCTTTTCCCAGGTACATTCATGTGGTAAATAGACCTAATGCAGTTTTTATCTAAAGCCATTTTATAACCAATAGAAATTAAATAGAGGAATATATAGAAGAGACCCAGGAGGGTTTTTGTGGAAAATCACAGACCAGTTGGTCTCGTTAGAGGATGAATTTTAAAGCAATTCTAATGCTTATACTCAGAAGACATTGCCCACCATTTGTCAAAACCTTAATTCTAAAATAACTCGTGGCCAAGGAGACACTGCTGTCAAATATGCAACCAAAATTTTCTTCTCAGTTTCTTCATTGGTTTGCTTGGTGCTCAAGTGCAAATAATGTAGTATTCCCTTCCTTAATATGGATGTGTCAATTTTTCTCATTTGCTTCAAAAATTGGAAGGATTTTTGCATTTTATGAGTTCTATAGTCCTTGGGCATGTATTACAGGCAGAGAGTACTCTATTTATAATACTGAGCTTTTAATAATGGTTGCTAAACAGGATCAGGCTTCAAATTATCAAGAGAGGAAACTTCAGTTCTGTGAGATAAGGGGATTAGTAAGGAGAACCCCAATTGGCAAGATTCTCCACTTTAGTAGCTTAGTAATAAAAAGGAGTTAAGGAAGGTGAGTGGAATAAATCGGTATGATCAGAGTTTGGTATGAAGTCAATGTTTAGATTTTGTACATACTATAGACCACCTGAGGACACAAATGATGGTTTATTATGGTCAAGTTTCACTATGGTCATGGGAACTACCTAACCCAGCACACTAATTCTCTGAGGGCTCTGGGTTTATGAATTAGCTCTTTTCTCTGGCAACTTTAAGCCACACAGTTCCCTCAATGGAAGATTTTGATGGGAAATGTTTATTGATTTGGAGGAATGACATTATTCTTCCAGGAAATTAGGCTGCAACCCTTCCTTCAGTCTCTCCATTGTTGACTGTTCAAGCCTCGAGGGATCCTAGAAACTCATTGCACTGTACAGCTCTTGTCTCCTCCTTTGCAACACAAACTCTCACATTCATACAGATGGGATCTAATGAATTGTACTACCTAATTTTCAGACAATGAAGTGCACTTATTCATTCAACAACCACTTACCAAGTATGTTCCAGTACTGGTGTGCATAGTAGCCTCCTTAGGTCACAACTTTGCATGTGGAAAAGTGAACTTAGGACAGCTTTGTCTAGCCCTGTGGCCAAAGGTTGGCAATCAACTGAACTATCCAGGTGACAAGGTTTCCTCTGACATGTTCCTGACCATGATAACCTATTTGCTGAGCATCAGCACGCTGTAATCTCTCCTCTCTCCTGGTCTTTCCCGGAGTCTATCTAGAGTCTCCTTTTTTCTTTTTCCCCACCATGCATACCTAGAATACCCCTTAGATTTGTGCCTTTACTTAGTGACTGAAAGGTCATAAAGACAGGGGCAGGGAAATGGGAGTAGGTAGAGGGAAGTGTTGGGCTCTAGATTTCCCAGAGGACCAGTTTCCAGCCTTCTTGCTCCTTTGGGGCAATGCCCAGTCTTTTCTAGGCCTCGCCTCTGGAAAACGCCGGGCTAGTCATGGCAGTGTGTTCCATTTCCGGTCCCCTGGCCGAGATATCTCACTCCCTGAGGGAGTCACAGATGATGGAGTCTTTCCTGGAGACCACGAAAGCCATCGGGGCTCTCTGCTGCTGGGTGGGGGTGCTGGCCAGCAAGGCCCCCTCCCTAGAAGCCCTCTTCCTCAACCCAGCAACCCTGACTCCAGGCATGGAGAAGATGAACACCAACCGCCGCCCACTAGTGAGCTTGCCCCTGGAGCTGTCGATGTCTCGGTGAGTTTGTGAATGCTTCAGACTCCCTCAGGCTGTCTTCCACCTAGAAGCCTTGAGCCAAGAAGCCTGAGTATCACCAATGGAAGGGATCTTTAGCCATCATCCTGTCCAATGCCTCCCCATTCTACTCCTTTTTTACAGACATAGAAGATGAGACCCAGAGAGAGAGAGAGATTGAGTTGGCCTGAGGTCAAACAGGTTTCCTGGTTTATTCTGCAGTAGTGATGCAAATTATATTCCATTTGACCCCATATTTCTTATCAGTGCTTCTCTGTCCCCACTCAACATCACTGCCTCAGTGATAGGCCTGCTTTCTTGTTGGACAGGTATGAGAAAAACTGAAGGACCCTTCTCTTCCTGCTTATGGCTCTATGCAAACTCTTCTAGGCTGGGCAAATGGCACCCAGAGAAATTCAGGAAGTTTCTTAGGACAGATCTGAATAGAAGCACAGTATTCCTTGCTTTACTCACAGAAGAAATAATTTGCCAAAATGAGCAAATTAGGCAACAGTGACTAACTGGCCAGCACTCTTTAATAGAATAATCGCAGAGTGTAATGGCAAGTCAGGGATTCCTCATTGGATTGGGAGCTGTGAGTTAATGTGAGCTCTCCCCTCCAGTTTCAGCAATGATGTTTCTAGTGTTTTTCATTTGCCCTCTCTGGCTAGCCAGGGATACAGCGCAATTGCAGACAGAATTGTAAAGAGACCAATAAATGGTTCCACTGATACAGATAAGTCTTAGAGTTGCCTTAGCTCAGTCTCTGACTCAATGTCATGTGATTCAGGGTCCCAGCAAGAAACAGATGGACCACTCCGATGGGCTAATAGAGAGAGTTTAGTAAAGTATTTACAGAGGTGTGGGCTGGGCTTAGGGAATGAATAAGGGACAGTGAGGCACCTAGAAACTAGTAACAGGGGGAAGCTGTTACATGCCTAGGCTGAAGGGATGAAGGAAAGGACCAGAATTAGACTACAGCAGCAGCTGCCCTCATGTGTCAGAGAGGCTGCCTGGCAGAACTTGTGATCTTAGTAGAGGATCACAGCTCTGCTACTCAGCCATAGCACAGGAGAGAAGAAGCCAGGGGAAAATACCTGACTTGTCTCTCCTTCTATCCTTGAATGTCCTGCCTATGTCTCCCATTAACCAAACCTGGCCAGAACCAGAGGGTAAGGATAACACATTTTGTACAGGTCTCCCTCCCCAGGAACAGAGACAGTGGAGAAGGTAGAGAGTAGGTCTCCAGGGCAAATGGAATCACCAAAACAGAGCCTTAAAGTCAAAGGCATCCTGCAGGGCTCGCTGGGAGTGCATTAGCACTCTAGAAAATGCCTCACTGAAGGCATTTATGCCTCTCTTGTGATGGGCCCCCACCCTCCTTTCTCTGTTCCTGAGATTATCATGTGTCTAGATATTCTGACCTTGATTCATTCTAGTTAAATTACCAGGCTAGTGAAAGATTGACTCTTTAGGCCCTTCTGTTTAGTATGGACCTATATTTTAGTCCCAACTGTGCCAGTTACCACCTGAGTCACATTTAGCAAGTCCTTTAAATTATCCAAGCCTCCTTATCTCCCCTTGTAAAAAGGTTTCTAAATAGCTGGAGGGTTAAATGAAATAACAAAGTGCCTACATTTTTATACATGTCTGACAGATAACAATAAATAGTGACTACTAGTATCATTTGCTTAGTCCTCATGGTTGGAAGCTTTATAAACTTTCTTTATGTTTATTAACTGTTTATACTTACAGTATTCAGCAATAACATGCTGTACAGATTTGTAGCCTAGGAGCAATAGGCCACACTATTTAGCCTAGATGTATAGAAGGCTATAGCATTTGTTTGTGTAAATACAATGTAGGATATTCACAGAATGATGAAATCACCTAATGATGTATTTGTCAGAACATATCTCTATCTGTAAGTGACATATGACTATATATGAGATTGGCCAATAAGAGGCTGAATGATCCAACTTTGCCATGAAGATGTCAGGTTAAAGGAGCGTCCTGACTCTGTTTGTGTCTCAACAGGCATTCGATGCAGGACAAAAGAAGACTTTCTTGTCAGCAGAATACTTAGATGAACCTTTCCGGGCCCAAAGGGCAATGAGTGTTGTCAGTATCATAACCTCCGTCCTTGAGGGTAAGTGCTGCTCACTGCATTGTTCATCCACTGTGTCATGATGAATTCCCATGTGAGCAGCAGAAGCAATGTCTCTAACTCGTGTCAAAGCGTCCATCTCTGTGTACTGGAATTCCTAATACTCCTCATTCTTATGTCTTAGGCCCCACCATCTTTTATCAATGTGGTTTTTTAGATGCATAATTTTTCAAAAACAAAATAATTTGATGAATAAAATATCCTAGCTCATCTCTTGTTCAAATTAGCATCTGCTTTGCATGCATCACAAAACTAGATTTTTAAAAGTGATCTGGCAGGCATCAGAGAATTTCAGGCCAGCTCTCCGGTGTACAAGAAATTTAACCATCCTTAATTTTCCAACATGGCAAAATTATCCTTTTCATGGTTTCATAATTATATTTTTCATGGTTGTAACTTACACAGTTGTAATTTAATTTCTTGTTCAAGCCCACATTATGCTGAATATTGGTAGACAATTTAAGTCCATTAATCAGTCAATTTAACTAAGTCCTTGTTAATTTAGAATTCTACCAATCAAGTGGGAATCCACTCAGTCTCCCCAGCAATGTATCTAAGTTCCAAGCCAAAGGGTAGAGGAAGAGAGTGGCAATGGTCATGGCCACCTATCCACACACTTTGCCATAGAGCGTGATTGCATCCCTCTAACCTTGAGCAGGAGGTCTGTTCTAATCCAGCCTTTTTTTGCCTTAGGGTATGGAGAATGCAACCTTCATCATTTCCAGTTATCTTACTCCCCTAGACAAATTAAACTGACCTAGGCTGGAGGAGTTGTAAGGGAGCTTGAGTGCTGGTGTGCCCCTGAGAAGGAGTCCTGTGTGTTTTCCTCCCAGCCCTAGGCTGAATCCTAATTTGAGGGGAAGAAGTGGTGAGCAATAGAAACCAAAAAAATGTTTGAGGGATTTCAAAACAGAGCCAATGTGTGCCCTACTTTCTAGGTAAAGCCAGAGGTGGCAAGCCTTAGAGAGAGATTCACATGCACAGAAACTGTAGAGTAGAACTAGCCAGTAATTTACCAGATATGCAGAGATGTATCTGTTTTGCTCACAGCTGGATCTCCATTACCTAGAAGAGCAGTGCATTAACAAAGGTGATATCTGGGGGGTGATGCTACTTTATAGTGGTGTAAACTGATAAAAAGGAAGAAAATAAGGAATAGTCAGGTATCTTGGGGTAGGATAGACTTCCGCTCAAGGAAGATACTGTGAGAGTAGAGTAAGAAAGGCAGGAGAAACAAAGACGTTTTTAAGAAAATCTGGGGAAAGGCAACCTTAGCTTAGGTCAACCTTAGGTTAGAGAAGGCCTGAAGCAGCCTCAGAGAGAGCCCTGTGGCTCCAGAATGGCAGAGAGGAGCTGGGTGATGACTTAGCCCCCAGTGAAGGCTATGGAAAAAGATGAGTGAGACCCATAGAGGGAAACTAGAGAGAGAAGAGCCATCTTCATGGACTCTCCCACCAGGGACCAGATGGGAGCTACAACTCAGGCAAAATACGCTTAGCAATGACAAAGGACCAGCTCAACTCTCCTCCAGGTTTGAGCCCTACAAAACTCAGACACAGACTCAGATATTGAAATAGTACCCAACATTGACTTAACAAGAGACAGTTCCCATGGTGAGGACTTGAAGCATAAATTGAATTCAACTAAGTTACATTTCTTGCATACCTGAGTTTGTGCTTTGAGAAATGTGTACTCATTTCTCAACAACAACGTGTACAGACTCTCTTCTATTATTTTTTCTCATTGATAATTATTTAATATCACTGTTCATTGTAATAGAGTCTGTACTTCTAACATTTTTCATGGCATAGAATTTGACCAAGCTGCTATTCCCACAATTTCTTTCTTGGCCAGATAGGTTTCTTGGTTTTTGTTTGTTTGTTTTGGTTTTTGTTTTGCTATTAGTCATCAAACAGCAAACATTGAATACAAACAGTTTGTTTTTTTCATTTATTCTCGAAAACTGTTAAGCTTCCTCCTTCTCTCTTCTTTCATTTATTCTCTTATCAACAAGCATTTGTTGAGCCCCTGTTGTGTGTGTGGCATTGCTTCAGGTGCTGGGATTCAGTGGTGAACTGATATAAGCTTGCGCTCATGGAATCTACATCCTAGCAGGGAAGGGGACATGAAAGATGAATAGATATTCAGATATAAGAAGTAGGAGAAAAAATAAAGTAGGAAAGGAGATGGAAAATTCTCTCCAAGAAGAACAGAAATTTGAATTAAATAAAGGAATCAGCTATGTAAGAAGCTGAGGAGAAAGTTCGCAGACACAAATAGCAAGTACAATGCATTCTGCAAGAATGAGATAGCAACAAGCAAAATGCATGTCCCAAACACATCCCAGTGTAGGTAAGACAAAAGAAGGTCATGGGATGAATGAGAGGCAACAGAGGGTAGTAGTTAGAACACTGCTCTGGAACCCGGCTGCCTGGGTTTGCATCCTAGCTCTTCCACTTATGGTACACAGTGTGACCTTGGAGCTTCAGTAACTTTTCTGTGCCTCAGATTTTTAGCCTGTAAAGTAGTGAGAATATACTTATCTTGTAGAGTTTTGTGAGGATTAAATGAGTCAGGTCAAGCAAAATGCTTAGAACCACGACTGGCATGTAGTAAATACTCATCACATTTGGCTATTATGATTTACTAGGTTGTAAAATCTATGAGGGCGGAGATGTGTCTGTTTTGCTCACAGCCTGACCTCCATTACCCAGAAGACAAGTGCATGCACAAAGGTGATATTTGGGTGGTTGGCGGCGGGAGTGATGCTAATTTATAACAGTGTGAACTGATAAAAAGGAAGAAAATAAGTAATAGGCAAATATGTTGGGGTAGGGTAGACTTCCCCTCAAGGAAGATGCTGTGACAGTAGAGTAAGAAAGGCCAGAGAAACAATGATGTTTTAAAGAAAATCTGAGGAGAGGAAACCTTAGGTTAAATAAAAGAGGTCAAAGTTTTCTGAGCATAGGGCACATCATCTTCTCTGGTTGGATGTTATATTCATTACACACATGAGACACCTGAAGGATGCTTCATGCTGCCAGAGTCGTAATAGGATTTATAAAGGCCACCTCTTTAAGAGGGATTATGCTTAAGAAAGTTTGGCCCAAACCAACAGGAGTTTGACAATTCACTGAACTCAAAGCATGTGAACCTTCTATGCTTGGCATGCAAGCTGAGATTTCACTGTTGTCCAAGCCAGAATTCCAGAAGCTGGGTCACAGCAGTGAGCTTACTGGAGTGGTGAGTGCCTTAAAGAGGAAGTGCAGGATACTAAGTGAAATATGGTAAGATTTGCCTGGAGCCAGGGAATTATCCCAGGCTGAAAGGACTGCAAAGCAGAAGAGAGAATAACATTTGAAGGAATGAAAAAGCCTAATGTAGCTGTAACCTAGAGAACCAGGAGAACTGAAAATAAGGCTAGAGGGATGAGTTGGGGCCAGATCCCAAGAAACTCCTAGACTGCATTAAGGATATGGGACTTCATGCTAAGAGGAGTGAGGTGCCATTAAAGGATTGGAGGCAGGGAAGTTACATTTGCATTTTATAAAAGTTACTCTGGATACTTGTAGATATAAGAGGGGAAGTATGTTGTCAGGGAAACCAGTTAGGAGGAAGCCAAGTACAAGATGGCAGTGCTTGAACTCTGGGGTAGTGACAGTGGGGCTGATGGGAAATGGAAGGATATGAGAGCTATCTAAGGGGCATAAGATTCAGGACTTGATGATGCATATGGTTCTGGTATAAGTGCCTTAAGATATGCTGATACAATAACTCATAAAGGGAGCATTTTAGGGGAAGAAGGGGGCTTTAATGGGGAAAGATGAAAGAAGATTATGAGTTGAGTCTGGGATATATTGGGTTTCAGGTGCCCTTGGAGCATCTAGGTAGGGGTGTCCAGTAGGAAGTTGACTTTAGGATATAGGGGTCTAGAACTTAGATGACAAAGATGGCATATATATATATATATATATATATATATATATATATACACATACACACACATATATATATGTATATATATATATTTGAAAGCAGTTTGAATGTAAGTGGTAATTTTGAAAAGATGCAACTGGATGAGTTAACACAGAAGGGTTGAAGTAAGAAGAGAAGAAGGCCTCAGACACAGTTCTAACAAACTAAATGTTTAATAGTTAAGCAGAAGAGATGGACCCAGCAGAGGAGACTGAAAAGGAAGGTACAGAAATAGGAAAAGATCTAGAAGAATACAATGCAGAGCCAAGAGTAAAAATTTCTTCAGAAAGAATAGAAAGGCCAACAGAGGCAATGCTGTGGAAAGGTCAGGTAAAATGAAACATGAAAAGGTCCAGGGGCTTTAGTCACATGGATGTCTTTGGTGATCTAAGCAAAAGAGGATTAATGGAGTAAAAGGGTAGGCAGAGGTCATATTGAAGGGGCTTAAAGAGCGGGTGAGAGGTGAAGAAATGGAGATATGGTGTACAGACAATTTTTTCAAGAAGTTTGCCTGTGAGGAAAAGGAGATAGTAAGATGGCACTAGGGAGTAGGGAGAATTGAAAGAAGACTTTTTGGTTTTTGTGTTTGGGGGAGTTTTTGTTTGTTTGTTTGTTTTGAGCATATTTGCATGCTAATGGGAAGGATCTGGGAGAATAAGAGAGACTGAAGATTAAGGGAAGAAAGGGTATATTCAACAGACTCCTGCAGTGGCAGGAGAGAATGCACAGTGGAGACACTGGTGTTAACAGGAGCAGGGGGACCTCTTTAAGCGTAACAGGATGAAGGGAGGAAAGTTCTCTCTGTTATCAATTTTATATAGGAGATCCTTTTGAGGTGGGGCCATGGTCATGTTTTAAGCTGGCAAGTAGATACTTTTAGTCTCTGTGTCTCTGCAATCAATCAAGTTCAACAATGTCAAGGCTGTTTACAAAGATGCCTCTCTTTTCCCATCATGCCTCACAGGTGGCTGCTTCCTGCCAAAATGGCACATCTAAAGTGTCCTCATTCATGACTACTTCCTGTGCTTCTTTTTAGTGCCAAAATAGGTCCAGGGAAGTTCTTGCTTTTGTTCCAAACAGGGATTGTTGATTTTGGTCCCAGGATGTGCCAACTTCTTTAGAGAACTATGCTTTGCCAGTTTGGACATCTGTAGTTTCTAAAATCCAGTCAGTGATTAAAACAGCATGACAGTGAAAATAAATAGTCTGATCTGTGGAAGAGAAATGGACATGTGTATATGAAAACACATGAACATAAATGGTAATAACATGACAAAAATGACATTTCAAATCTTGGGAAAATGAAAAATTATTCAATAAGTAATGTTGATATAAATAATCAGAGACAGGAAAAAATAAACCACATCAATATCTTACTCTTTACCCAAAATAAATTTCAAATGCACCAAAGTTTTAAGTGAAAAAAGTGAAACTACAAAAGAAAGAAAAAAACATGGAGAAGGTATTTTCCAAGCCTTGTAGTGAGAAAAGCTTCATATGTAAGGCATGTATGCATATGTGGCTAACCTGCATAATGTGCACATTTACCCTAAAACTTAAAGTATAATTTAAAAAAAGAAAAAAATTTAAAATAAAAAAATGAAGAAAATTGAAAAAAAAAGAAAACAAATAAAAATACTACTGATAAATCTTGACTACATACACATTTTTAAATTATCAGGAAAAAACACTGTTAGCAAAATTTGGGGGCACACAGTTATTTCTCTAAACTGATTTTTCAAGTGCATATAGTAAAAAAAACCCTAAACACTGCAAGTGTGTATGCAATGACAACTATGTCTCCTTCCCACTATAGACCCTTCCTGCTCCCCAGACTCCTGGTGCACCCCCCTCCCACTCCAGTTTATCTTATATTCTTCAAGCAATGTTCTATGCATATATAAGCACATGCATGCACATACACACACTACACATACATATTTTTCTACATCACCACAAATAAATCTGCCTCATTCTTTTTCACAATGTATTATGCTTTATCAGTTGTTGTTTTCCATTATTATTATAAACAATGGACATCATCTTTCTTTGCCCACATAGGCTGATATCTGTTCAAGATGCATGTCTTGCCGAGGAATTTATAGATAAACCTAAAGCTAAGAAATTGCCCTCCATCGAGGTGAACACAGTTACACATCCACATGGAGATTTAACGGAGGCAGGTAGAGAGATAAGCCTAGTGTTCAGGAAGGGATCAGAAATTGGACACATAATTTGGTGGGAATTGGCATAGAGATGGTTTTTTAGGGCTATGAGATTCAAAAACACAGAGAAGCTAAGGTGAGAATGCTTGAGTTATTCAACATTTACAAGCAGGAAAGGAAAAGGGAGACAAAAACAGAAAGTGAAAAGAAGTGGAGAAATATGGGAGAAAAAGCAAGAGATGATGCAGTCCCCAGAGCCAAATGAACACTGGTTTCAAAGAGAACAATATAAATTCTGTCAAATGTTATAAGGGTAAGATGAGAACTGGAAATCAATTATTTGAATTTGGAAGCAGTTAAAGTCAGATTCCAGGGGCTGCAGAGAAAAAAAGAGGTAAGGAGCTGTAGAAGGTTAAGTGTAGAAAACTCTTGCAAGGAATTTTACTGTCAAAGAAGCAGTAATGAAAGAGAGCATGGGGTCCAGAGAGACTTTTTCAAAGCCGCGAGATAATTAAAGCATGTTTACCTGCTCATGGCAATGGTCTGGAAGGGAGACAGGTCTGGATGATGCAGGCCTTGAATAGGTGGGAGAGGAAAGAGCCCGGCACAAGTGGGAGGGGTAGAGCCACCCACGCACTGTGTGGGGCACGAAGACAGAATACCAGGTGGACTTGCAGATATGCCAGAGATTTAGTGGTAGGAAGAAGGGGAAGTTCTTTTGTGAACTAGTCTATATTCTGAGTGAAATAAAAGGGAGCTCATCAACAGAGAGTAAGTAGAAAGAAGAGCCGGAGGTTAGACGGGGGAAGAGGAAGTGAGAAACAGTCATCTGGAAGAATGAAGGAATGGAATATAATATAGAAACACAGCCAGATTTCTGGGCAGGGCAAGGCTGGGGCTCACAGATCTCTAGGAGGGTTGGTAAGCATAGTTGTGTATTTTTTCCAGCCAGGTCCAGCTGGTTGGGTGCAAGTACAGAGTAGTCTACAAATTGGGTCTATTCACAGTTCAGGTTTTGCCAGGCAAGTACAATGAAGGAGGAGAGGGGCAAAGGAATTGAGGGTGCCTACAAGGGAGTAGTTAGAGAGATGGATGTGAAATCTAAGCTGGGCAAATTGAGAAGTAAGGACATGATATAGGTGATGGGCAGTAAAAATATGTAATGTCAGCAGTTTAAAGGACTGGATGGGGCAGATATTAATTGGAGTTGCAGGACTAAAGGAGTTCAAAATATAGGAAATGAATACCAGAGACAGAGAGAGGGCTGAAGTCAAAATGTTGGAGGTGGTACTTATTATTAACAACAAGGTCTAGAGGATGACCGCAGAATTGGGGTCCAAGGTGACACATGGCTGACAGCTGTCATTGACCACACTATAATGCAGAACTCGAGGAGTCTGAACAGAAGTGCCCACCCTGCTTGACCAGCTTGTCTCAGAAGTATCTGATCTGGGATTGCTGCCCCATGTGGGTGAAGCTCAAGACAATTCTCTTTGGGCTTGTGACGGATCCCTTTGCAGAGCTCACCATCACCTTGTGCATCGTGGTGAACACCATCTTCATGGCCATGGAGCACCATGGCATGAGCCCTACCTTCGAAGCCATGCTCCAGATAGGCAACATCGTGAGTGCTCTGGCTGGTACTGCCCTCACTGGCGTGGTCGGGGCGGGGTGGGGATGATGGCAGGGTGCAGTTCGGGTGGGGTGGGGCATGCCCTTATTGGTTAGTGTGTTGAGCCAGTTAGCTGAGAGCAGAGCAGCAGTGCAGGAGACATGTGGACACTGGGTTCAGGAGGTCAAGTGTGGGAGGACCCAGGCTGAGCTGGTGGGAATGGCTTTGGGATGAACAAGCACAGGAAAGTGAACCAGGAGAGCCTGGAGCCAGAGGCAGCTAGTTGCCCTTTACAAAACATAGGACAACCACTTGAATTTTCTGGATCTCAGTTGCCTCTACCTTGAAATGAGGATAAATGTCACTTTTTTATCTAAAGACAATCTTTGGACTTGTCTGTTTCCTTCCAATGTAATTACTTTTATGACCACAATTAACAGTAGCCACGATTTTCAATGTTGTCACTTAAGGAGAAAAAGAGTTGGGGAGGGAAGGGGAAGACAGTGGAAGGGAGGAAACATGGAAACAAGGTTGCTGTTGTAACAAAAGCATCAGGAAGTCGGAACTTTTCTTTGGCATCTAGTTTAGACATCATGCTGCTTTAAATTTTCCTCCATCACTTGTTAGTATTATTTACTCTTAACAAGATACAATCCATACTGGATTTCACTATATACTGGATAAAGTATCTGACTCAGAGGGACAACAACTGCTCCAACCTTGCCCCACCCACAGGAATAGTAATAATACTCAGATGCTTCTCATGGATTATCTCATTTTATTTTCACAACAACCCTACATGGTAGGCACTCTTGTTATTCCCTTTCCCAGATGGAAAAATGGAGATGCAAAAGGTTAAATAATCTTCCCAAGATCATAGAGTTGAGTTTAGTGGCCACATCAGAATTTGAACCCAGACAGACCGGCTGCCAAGCCCGCTTTTCTTAACCCCTGAATTAAGCCACCTCACAAGACTTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTCAAACACACGCACACATGATAGGCAGAAGCTAGAAAGGGTCAGCATCCTGCCTTCATGTCCGCAGCAGGGTCCACCAAGCTGGAGGTGACCTACTGGATTCACCAGAACTGTCTGGCTTTGGGGTCCAGCTTCTTATAGGCTGCAGACTTAGGTGGAGGGGGAAGGCCCAGAGGAAATGTGACTACTTGTCCCTAGAAACCACTCCCTCCGCCATAATCCAGGGCTGTGAAAGGGGCAGCAGTAGGTTCTGGAACATGGTGGAATCAGCATGCCTCATGTTTTGGCAGCACCTAGGCTGAGGTCACATCCTAAAGTTGATATGAAAAGGAAAAATGGAAAGAGAAGAGAATGCTTATTCCCAAGAAGTCAGCCTAAGACCACAGAGGGCCTTATGGAATAGACATTTGAGCCCACACCCTCATTCTACAGTTAGAAAAGTGGAAGCAAGAGAGAGAAAAGGCCTTTCCCACAGTGTCAGAGGAAAGTGCCACTTTTTTAAAGCAAGAAAGTCATGTGCTTGGTGCCTGCAATGAGTCAGGTGCTTTATGAATGTTATTTCAGTGTCACAACAACATTATGAGGTTAACATCACCCTCACATGACAAAAGAGAGCACTAAGACTCAGAGAGGTTAAATATCCTGCCCAAAATTGTATAGCTGGTGAGGGTGGATCCAGGGTGGACCCAGGTCTCTGGAAGACACAAGACCTCAACCCTACCCCCCACCACACCACACTGACTTGCCTACAAGGGACACTCCATAGATACTCTTTGAATCAGTCTTGTGGCATGAGAAAGTCACTTAAACTCTCAGCCTGTTTTCTTCTCCATAAGAAGGATGCATTGTCTGTTTCTCAGGCTGGTGAGGGGCTCAGATAAAAAGATATGAAGGAAAAGCACCTGACCCTGGTCGTTGCTTTCCTCCTTCTCTGTCTCCCTTCCCTGCTGGTTGTTGTGTTACCCGAGCCTGGTTCTGACCTCTCTCAGTGTCAGAATTCTGTCCTATTTAAGACAATTAGAGACACACAGAGCCTAAAAGAATCTTAGAGGCAATGAATCATTGTTCTTACTAGTCACTGTTACTTATGAATGAATTACTGTCTGCCAGGCCCTTTGTTAAGAACTGCACATGCATTGGCTCATTTTATGCCCATTATACAGATGAGGAAATTGAGGTACAGGAAGGATGCATTATTTGTCCAAGCTTACATAAGCTAGGAAATGTCTGGGCTGGGATTTTAATCCAGGTCTGTGATGTCCCACAACCCACACGTTTTAACTACTGAACCAGACTGACTCTTCACTGACAAAATGATATTATAATAATGATCTTAAAGACTATCCGCTTATTCCACTCCCACCACCATTCACGTGGAAAAACTGCAGCATGCACAGCCCCTGAGAATGGTGCAGAAATGTCTCCATTCTGTCACGTTGACTGTTACCCTGGCCAATCCTCAGGAGCCCACAGATAACATAGCAACTGAACAAAACAAAAGGATTTTCTATTTTGTAAATAAGACATTTTCTTGCTGCCATTTTTATCATTGCCTCCGACCCCACAGATCCCACTGTGATGAAAGCTTGTTTCCACTCCTAGGTCTTTACCATATTTTTTACTGCTGAAATGGTCTTCAAAATCATTGCCTTCGACCCATACTATTATTTCCAGAAGAAGTGGAATATCTTTGACTGCATCATCGTCACTGTGAGTCTGCTAGAGCTGGGCGTGGCCAAGAAGGGAAGCCTGTCTGTGCTGCGGAGCTTCCGCTTGGTAATGTTTTTCTCTTGATGGCTTGGGGAAACTCCCACCCAGATTCAGGGAAGACACTGTGCTCTGGGGACTGCACCAGCCAGCTCCCCCTTCCTCTCCTGTCCTTGTGGGAGGGAGCAGGGTCTGACTCCTGGGCGACTGGAGACTTGGGTCTGTGGCCCCAGCTTTTCGCTGACTCAGAGTGGGGACATGCCTTCTCCTTTCTGGGCCTTGGCTCCCTTATCTGCACCAAGAGGGGACTTCCAAGCACTGTCCTCGTCTCTTATCAAATAACAATAACAGCAATAATCACAGCAGCTAATACTTACTGAGCACTTACAATGTGTCAGGCATAGCACTGGACACTTGAAAAAACGCACCTCATTTTATGCTCACAACAACCCTGAGAGATAGTCGTTTTTAAGATCCTCAGGGGTGGGTGAGGAAAAAGAATGGTTCCATGATGACCCGAAAGTCTCCCATCTGCCAAGCAAGCGAATAAGCCACAGACCTGGATCTCAAATCCAGGTTGTCTGACTCCAGAGCCCCAGCTCCTTCCAGCCTCAAGGAAGTCATTCTCTGATTCCCTTGTGCCCGAAATCACAGCCCCAATGTCTCAAGCACCTTCAACCTGCCTATTAGTCTCACTCACATGCAATGATGGCTCTTCTCTAAACTCAGCTCCAGGACGCCCTCTACAGGAGAAGTGCCACAGGCCACCCACTTTTATCAGAGCCACAGGTTTGTCAGCCAAACCTGTCAGATTCCCAGGAGTGTCGGGTCACTAGGTCTCCAGGCCATCTGGAGGAAGCTTGGCATCTCACTGACTCTTGGGAATAGGAAAGAGGCCTGAAGAAATGTCACGGCTTGTTAGCGCAGGCCGCCTGCTCTCAGGAATCTCTCAGTATTAACTGGGAGCCCTGCATCTAGACTTGAAGAGAGGTGACAAAACAAAGGGGAGTATCCAACAATCTTCCCAAGGTCCTGACAAGTGTTGGCATCCAGAGGTTGAAGGGGGTGCATGGTTCAGGAGGCAACGAGACTCTAGGACAGATCTGCTTGGGACCCTATCACAGGGGCCAGTGAGACAGTGCTGAGCCCCTGAACTTGGCGTACCTGGATTAGGGGTGGCAAACTCAAATGCCCATGTGCAGTGACATAGAGCCAGCCATGGAAGAGATGTGGGCACTGGATATTTGCCTCAGGTAGACAGGCAAAGATGACTCCATTTCACCTAGGCAGACCCAAAGCAGTGCACAACTCCTGGGAGGGATGTTTACATAGAAATCAATGTGACCATCTCAGGCCAGGTGCAGTGGCTCACACCTGTAATCCTAGTACTTTGGGAGGCCGAGGCAGGCAGATCGCTTGAGCCCAGGAGTTCAAGGGCAACATGGTGAAACCCCGTCTCTACAAAAAATGAAAAAATTAGCCAGGCATGGTGGTGCACATCTGTTATCCTAGCTACTCAGAAGACTGAGGTGGGAGGATCACCTGAGACCAGGGAGGTGGAGGCTGCAGTGCACCATGATCGTGCCACTGCACTCCAGCCTGGGAGACAGAGTAAGAACCTGTCTCAAAAAAGAAAGGAGACACAGAGAGAGAGGAATAAAGATAGAAAAAGCGAGAGAAAGACAGATGAAAGAAAGAGAGAGAGCAAGAGGGAGAGAGAAAGAAAAAGAGAAAGAAAGAAAGAAGAAAGAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAAAGAGGAAGGGAGGGAGGGAGGGAGAGAGAGAGGGAGGGAGGGAGGGAGAGAGAGAGGGAGGGAGGGAGGGAAGGACGAGCTATGGCCTGTCCATTATTGTCAGAGTTACTAATTTTCCAAAAAAGTCAGAAATCCAGATTTTTGTGCAAATGTCTTTAATATTGGCTACTAATCCAGTTTTTAAAAACACTCTAAAGACCAAATAAAGCATGTCCTGAGACACAGCTGTGCCATGAAACGCTGGTTGATGGCATCTGCCTAAGCTGATGGTACTGTCACCATCCCAGGGAGGAGAAGGGTAGAGTAGGCCTGGTAGGACTCTTTATCCATAACTTGAAAATCTTACAAGGTCAGAATTAAATCACATTTTTAGAAGGACAGATTTCTCTAGGTATCTAGAAATTGATGTCAAGATGTAGGCCCCTAAGCTAATGGTCAATATAAGGTACCTATGCACAATCGTCAAGAGAGCAGGAGATATCACACCCCCATACCCTGGCAGGCCCACAGTTAGTTTTCTAATAAAATACGAGGCTGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGATCACAAGGTCAGGAGATCGAGACCATCTTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCACGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCGGAGCTTACAGTGAGCCGAGATTGCGCCACTGCAATCCGGCCTGGGCTAAACAGCGGGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAACGAACATTTCTGCTACACTTGGGGAAGCAGAAGCTTAGTGTTTTAAGGTCTATGTGCTACTTTTGTTCTGATCCCACAGTCTAAAGTCCCAAACAACCAAAAATAGGCAAGTTTACTTCACAGTCTGCCCCTACAGGATTCTGCAGGGGAATGCTACCCTGTCCCCACCTCGCTGATTTAACATGCTGATCAGTGCTCAATTCCCTAACCACACCCACCAACTACACACTTTCTTGGGATCTATTCCACTTCCCTCATGACCTAGCTTAGATTTCACAGCCCACCAATATTATTATGCCCTTGAAAACAAACTCAATCACCTTTTCCCTCTGCCTCTTTGCGTTTTTGTGTTTGCCAAAACCTCACCGGAGAGTCTAAGTCAAATATTTCTCTCCACCTTGTCTGCACCTGAGCTGTGAGAAATGCTGGAGAAAAATCACCGCCAGCTGGTCAGTTTCCTGGTTTTAATTAATAGACTTTATTTTCAGGACAGTTTTCAGTTTTCAGAAAACTGGAGCAGATTGAACAGAAAGCTTTCATATACCATCAATCCCACAACACACACACACACACACACACACACACACACACACACACACACAGAGTTTCCCTATTACCAACATCTTATGTTAGTACCTACATTTGTTACAAATAACTTCACTGGTTTCCCATTGCTCTGAGAATAAAATCCAGACTCCTCATGATGGTGTACAAAGTTCTGCATAATCAGGACCTGCCAGTTCACCAGTCTCCGTCTTGCCCACTTTGCTCCAGCCAAAAAGGCCTACTCTCTCTTCTTCACACATAACAGAGAACTTCTTGGCTTTGGGCCTTTGAAGTTTTTTTCGCTGTGCCTGGAATACTTTTCCTTTAGTCTTCACTTAGCTCACTTATTCTCAAACTCTGTGTTCAATGCACTTGTCACCTCCTTAGAGGGGACCTCCCCAACCACACTATCTAAAGTTGGATTCCTTCATCAAATTAACCTGGCATTTACAATTGTCCTGCTATCCCATGAGGCAAAAACCAGAGTTTCTTGTCTACCGTTTTATCCCAAGGGCCTAGCACAAGGCCCAGCACATACATAGGAACTCAATAAACACCTGTTGATTGATTGAAGGAAGGAAAGAAAGAGAGGAAGGTGGAGGGAAAAAGGAAGAAATAAAAAGTCTGTCTTCCTTCTCTCCCAAAATAGCATTGCTTCTCCTCCCGTCACTTAAAGCCTTGCTCCATTTGCTCTGTTAGTCCATTGGTCTATCCTGGCACCAATATCAGTCTTAATTATGGCAGCTTTAAATATATCTGTGTCTGTAAATCAAGTAATCCACCTAATTTACTTCAAGAATATCTTGGCCTGCACACTTTCATATTCACTTTAGAATTATCTTGCCAAGTTACATACCCACATACACACAAAACTGCTATTGTTTTTCCCATTAGGGACTGCTGTAATTGATAGGTCAATTTGGGAAGACTTATAATCTTTACATAATTCAGTCTTCTGATCTATGAAGATAGTATACGCTGTATACCAGTCTTTCAATAAAGTTTAAAAATGTTTCCATGAGGTTTTTATAATTTTCTCCATAGGATCTTGCATATCTTCTTAAATGTTCTTTTCTGGCATTTTTGGCTAGCATTTTGATATTTAACATTTTTGTATCTAGAAATGTCTCTTATTAATTCTAATAATTTATCTATACAATGTTTTAAATTTTCTAACATAATCACATCATGTGAAAATAACAATGAGTTTAATTTCTTCTTTTCTTTCTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCACCCAGGCTAGAGTGCAGTGGCGTGATCTCGGCTCATTGCCAGCTCCACCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCACCCACCACCATGCCTGGCTAATTCTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCATGATCTGCCTGCCTTGGCCTCCTAAAGTGCTGGGATTACAGGCATGAGCCACCCACGCCCAGCCTAATTTCTTCTTTTCTAATCCTTATGCCATTAGGTTCTTTTTACTAGATCACTGTGCTAGTTAGGACATCCAGTACACTGTTGAAAAGAAGTGTTAATAACAGGTATCTTTATCAATTTTCTGATCCCAAAAAGAATCCTTTCAATATTTCACCACCTCTAACAATTGTGGCAGGCTTTTTTTAAAGATAAGAAATCTTTATCAGTTCAGGAAGTTCTCTTTTATCAAGAATATTTATCATGAATGGATGTTTAATTTTATCAGACAATTATTCTACATGTATTGAGATAATCATGTATTTTCTCCTTCAATCTGTTAAAGTGGTGAATTATATTAATTGATTTTCTAATGTTAAATCAACTTTACATTCCCAGCTCAAACCTAGCTTGATCGTGATGCATTTTTTAATACATTGGAAGATTTAGTTTGCTAAAAATTTGCTTAAGATTTTTTTATCCATATCAATGAGTAATATTTGATCTATAATTTTCCTGACTTTCATTGTCCTTGCCAGGTTTTGGTATCAAGGTTCCAAGATTATGCCATTTTTTTTTTAAAGCCAAGGGGGCCAGGTGGGGTGGCTCACGCCTGTAATCCCAGAATTTTGGGAGGCCAAGGTGGGAGGATTGCTTGAGGACAGGAGTTGGAGACCAGCCTGGCCAACAGGAGAAAACCCCATCTCTACTAAAAATATATAAATTAGCTGGGTGTGGTGGCACACACCTGTAATCCCAGCTACTCAGGAGGGTGAGGCATGAGAATTGCTTGAATCCCAGAGACAGAGGTTGCAGTGAGCTGAGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGCGAGACTCCGTCTCATTGAAAACAATAAAAAGAAGGCCAGGGGCAGTGGTTAATGCCTGTAATCCCAGCACTGTTGGAGGCGCAGGCAGGCGGATCACCTGAGGTCGGGAGTTCGAGACCAGCCTGACCAACATGGAGAAGACCCATCTCTACCAAAAATACAAAATTAGCCAGGTGTGATGGTGCATACTTTTAATCCCAGCTACTTGGGAGGCTGAGGGAGGAGAATTGCTTGAACCCGGGAGGCGGAGGTTGCGGTGAGCTGAGATTGCACCATTGCACTCCAGCCTGGGCAACAAGGGCAAAACTCCATCTCAAAAATAAATAAATAAATAAATAAATTTTAAATTTAAAAAAGAAAAGGAAAGAAATTAGCTGGGCATGATGGCACGTGTCTGTAGTCTCAGCTATTTGGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGGTCGTGGCTGCAGAAATTACAGCATAAATTAACTCTCCATTAGTTAACTTAATCAAAGCCAAAAGTTAGCTAAAATCTTTAACCTTCCTCCAAGACAACTTTTCATTCCAGATATCCTTTCCCAATTTATTGTAATGATAAGTATTTTAATTTTATCTTGTTTATAAAAATTTTTATTAATCAATGAAAAATAAAGCTACCTATTATACAGACAATCTCTAAAACTGAGTAACTGACCCCAATAAAGGTTTATTTCTCACTCGTATCATAGCCTAATGCAAGCCAGACAGCTCTAGGTGGGACTCCAAGAAGTGACTTTGCCATGCCTGAAGGAGGCCAATATAACTTCAAAGTTTGCCTCCTGCTTGTGGGAAAATGGAAAGCCAAGGTTTGTTTTAAGGTTTAAACAGTGAAAGGCTTTTTCAGTCAGGGATTTTGATTTATTTCACAATACAATTACTTCAAAAACTTAGATGGCTTCAGACCTCTCATTGCCACACAAAGTGCTTTCTTTGACAAAGTTCTCAATTGTTCTTTATTTCTTTGCTTTCCTTTCCCCATGCCTTTCTCTTGCACAAGAAACAGTGGCTACACCATTACTACAAGCTTTGCTAAGAGTTGTTTCATTTAATTGGAAAATTTAACTAGTCTTCAACATTTGAAGACTTAATCCCATTGCTCACTGCCTGGAATCTAGAAGCAGTACAGTTCCCTAAATCGTCAAGGCTCTAAACTTCTGGATTATATTTTCTTTTATTCTTATAAACCAGCTAAATCTTTCCTGAGCTAATCTCTTTCTTGTACTGTCTTGACCAACATAGCCAATTACAACTGACCCACAATACTAAGGTTTAATAAACCCTTCCCCTAGGGTTGCTGGTTCATGAGGCTCGTGCTCTGCCTCAAAATTTTTGCCAGCAATATTTTTGTCACTGAATAACATAGATTGACATTCCAAGCCTTCTCCTATCAGCTTCCTTGTTGCCCACCACATGATCATTAAGCCAGTGCCACATAGTTGATGCTTTTGAAAAGGCAATACCTTTTTTCAATGAAGCAATTTCTCCATTACTCAAGATAAGTACAACTAGCTGCAGTGATACCCTCTAAATCTGTGAGTTAACATAATAGAGATGTATGTCTCACAGCACATTCAGTGCTTGTTGGGAAGCTCTCCTGGGAGTGTCTACTTCAAGTAGCGCCTCAAGGATCTAGGATTATCCCATATCGTGATGTCACCATCTTCAGTTAGTGACCTTTATGGTGAACATGAAAGGGAAAGAAAAAGTTCAGGGGGTTTAAGGCTATGCCTAAAAGTCATGTATATTACTTCTGTCTCCATCTCCATTCCACCAGACAGAACCCACTAACATGACCCCAACTATTTGCAAGCAAAACTGGAAAACAGTCTTCCGTTGTGTCTGGGAGGAGAAAAGTGAATGGAGACTATATGACCAGTCTCTTCCACATCTCACAAGACATTGTTGTTGTGTCCACTCAATGTTTATTTAGATTCACTCTCATGCTTAGCACTTTCTTGCTCTTTATACCTTTGTTATAGAGCCAGTGGGGGCTCACTGCCCAATGCTCTAGAAGCCAATACTATGACTTTGGAAGCCAAGACGAGGACACCAGGCTTTTGAGAAAAGAAAAGCTTAATACTGCAAGCTGGCTGGCAAGAAGACAGGAGGTCAAGCTCAGATCTGTCTCCCTGTGCTGTCTTCAAGGCAGTATTTTTATTAGAAAATGTTTAGTGGGTGGATACCGGGATTTGCAGGTGATTGGTGGAAGGAAAGGGGAGGTTTGGAGAGTCCATGGGCCTGCACAGTTATCTCTTCATGCTTACTCATGGGTTGCATATGCAAATCTGGGGGGAGTTAGTATGCAATATGAAGTGGAAATTCAGTCCATGACGTCAGCAAGCTCATTCTGCACAAACTCCAGTCGGCCATCCTGGTTCCAATTGATTTCAGCCTGTTTTTTCATCATCACACAAGGGGAGGGAGTTTCAGTGTCTCAGCAAGTTGTTTCTTTACCTATCTGCTATCCTAAAAACTCAAGAATTTTTGTAAGTTACTGATTTCTTACTCTTTGGAACACAGTTTGAGTTTCAGATTTTCAGTGAGTTATTTCTTATCTGGTATCCTGTAAGCCTAAAAATTTAGTTATTGATTTCTTCAACCCTTTGGAGCACAGTTTCACATTCTTGTATATTACACATTACATATTACTTCTGGGATCACTTTCTATCTACCTGAAGTACCTCCTTTAGAATTTCTTTTAGTAAGTGCCTTCTGGCCATAACCTCTTTGTTTATCTGAAATTTGCTCTTCAGTTTGCCCGTGTCTTTGAGAGACATTTCTTATGAATGTAAAATTCTAGTTGATACTTTATCTTCCTTCAGTGAAGATAGTATTCCAACAATTAGCCTCCATTGTTGCTAATGAGCAGTCAGCTTTCAATTTAACTGTTATTCCTTCCTTTCTGGATGCTTTTTAAATCTTCTATCCATCTTTGGTGTTCTGCAGTTTCACTGTAATATGTCTAGGTGTGGACTTCTTTCCATTTATAATGCTTAAGATATACTGACTTCTTGAATCTTTAGTTTTGTAATTTTCTTCAATTCTGGAAAGTTCCCAGACATGATGTGGGCAGTCATAGCCTCCACGCCATTCTATTTTTTCTCCTTCTGGACTTTGGTTAGATAGATGTTAAACTCTCTCATTCTATCCTCTATGTCTCTTACACTATTTTGTTATTTTTCATCTTATGTCTCTCTAGGTTACATTCTGAATAATTTATTCTGATATATCCTCAGTTCACTAATTCTCTCCCAAACTGTGTCAAATCTGATGCAAACCTTCTTCATAATATTTTTAATTTTAAGTATTATATTTTTCATTTCTAAGAGTGTATTTTTCACAACTTGTTATTGTTATCTATAGTTTTTTTATTCTCTGCACATAGTTTCAAGTTATTATTTTATTTCTTTAACATATTATATCTAATTATACTGTTATCTCTGACTAATAATTCTATCATCTGAAGAAATGGAATGACTATTTCCACATCTGTGGTTTCTTCAAGATCTCATTCACTCTTGATGACTCATTCCTTCTGTTTTTGTTTGTTTTTTTACTGCAACTCTTAAACAATTTTTGAGGGAATAGTTTGAAACCTGAGATAAAGGCAGGTTCCTCCAGAGGTATTTGCATTTAATTCTGCAGAGCACTTTGTAGAATTGCTCTAAATTAAATTCTCAACTTAAAATACCTAGGTAGTGTGAATTCCAGATAAAATCCTATATGAGCACCACTTTGTTGTTTCAAATGCTCAGCGTCAATTTTCCTCCTGTAGCTGGTACCTAACACCAGCCATTCAATTTTCCTTGCATTCTCCTGGGATTGTTTGTGGTTCATCCTTACATGAAGGGTATAGCTCTTTGGGATCTAGTCTTAAAGCAGGGAGAAATTCCTGCTAGGCTTACAACCTTCGGCAGGCCATGGACTACGTTTCCTGCTCCCCATACTAGTTACTAATGCTCAGTTTTTATTTGTGTTTATCAGCTGCCTCTGTGACCAGTCCACAGAGTTGTTGTTTTAAGCTCTGCTTCCCATATGAGGAGAGTAACTTCCAGATTGGGTGTAAGGTGAACGTGGACCCGCTCTCCCCCATCACATAAAGAGGTGCATACAGCACTTACTGTATAACAGGATTAAACCTCCTCCCGTGGGATTTACTCACGGATATGGGTGATAAAATTGTAGCTACACTGTTGTGTTCAGCATTGCATCCTAGCACCTAAGCCACAGCCTGGCTCTTAGTAGGTGCTCCATAAACACACCTTGACTGACAGACTGACAGTTGAGCTCAGATTTTTTTTCCTGACAAATTAGAGAACCATACAAATATATTTTAAAACACATACATTTTTATGCTTGATGATTTTGCTGTCTTGTGGGACTTAGCATATTTGAGTTTACAGTGAAGACTTTTGAGACTTCATCTTTTCTCAGTAACTAAACCATTGGTTCCATGAGAGCAAGAGCCCACACACCTCTCTTCATACACCTCATAGGCTAGCACACTGCCATGCACCTGCACGGCTGGAGTTCTGGGGGGTAGGTTCGTTGGTCAATTAAATCAACTTATGAATGCTGAGTCCATGAAATCATGGACATTTGCAGTTTCAAAGCTGGTGGGAGCAACAGATGTTGTCCACTGCCCCAAGATGAGAAATGCCCTAAGAGATGTTTTGCCAATACCTATCGAATGCAGTGGGTCATCCCCTACCCGAAGGAGGCTGGTCTCTCTTCAGTCATAAATAAGCTTGTTCTCACTTATTTGTGAGCCCCATCACACCCACCCCACCTCCACAGAAGAGGCCCATATTCAGGAGGGATCAGGCCTGTGGCTATAGAACAGAGTACAGATCGCAAGGCCACCACCCCAGTAGAAGACCCTGACTCAGCCACAGTCAATGAAGCACATTGATACTTATGAACCATAGAAGGCAGATCATATTTTTGAGTAATTTATAGCATAGGAAGAAAGGTCTCCAAAAGCAGTGTCCAGAAAACGCCCTGGTTTAATGGGATGAGAGGCCTTGTGTTTTTCCCAGATCTTTGGTTTCCAGGCAAAACTTTAGATGAAAGGTAATGAGCACAGCTACCAAAACCTGACGGTCTCTGTTTCTCTGCAGCTGCGCGTATTCAAGCTGGCCAAATCCTGGCCCACCTTAAACACACTCATCAAGATCATCGGAAACTCAGTGGGGGCACTGGGGAACCTCACCATCATCCTGGCCATCATTGTCTTTGTCTTTGCTCTGGTTGGCAAGCAGCTCCTAGGGGAAAACTACCGTAACAACCGAAAAAATATCTCCGCGCCCCATGAAGACTGGCCCCGCTGGCACATGCACGACTTCTTCCACTCTTTCCTCATTGTCTTCCGTATCCTCTGTGGAGAGTGGATTGAGAACATGTGGGCCTGCATGGAAGTTGGCCAAAAATCCATATGCCTCATCCTTTTCTTGACGGTGATGGTGCTAGGGAACCTGGTGGTGAGTGGTGAACCCTGCTGGGGGCACTGTCTCCTGGGGAAAGGAGAAGGCTTCTGGGTTATCTGAAAAAGCTTTGATCTGCATTTTTATGCCCAGATTGTACTTCTCTGGTTGAAAAGTCTTCAAACTTTTCAAATTCATGTGGGATTCATTACAAGAAGTGTGCCTTGGGAGGCAGTCTGACTTGCAGGAATGAGAACATATGATTAGAAAAGAAAGACCTGGTGTAGAATCTCTGCTCCACCATTTACTAGGTGGGTGACCCCAGGCAAGAGACTTAATCTCCTCAAGATCTAATTTCCTTAAAATCAAGAAAATAATGCTTACTCATGGGTTTACTGGGAGAATTAAATGACAGGAGATGACATATGCAAATTGCCTAATGCAATGTCTGGGCTGAGGCAGGAACTCAATAAACGGTGTCTATTAGCTGTTAAAGGAAAAATGTCCTTCCCCAATCTGAGGGGGCTCAGAAAACATTTTTATTCAGGGAAGTAAGAGCTGACTGATTTCTCCAAGCTGCACAGGTCCTAGCTGGGAGCAGGGGGAGAGGCTGCAGGGAGGGGGCACAATGGGTCACGGACAACATCCAAGGCCACAGAAATAATAACAAAATCTACTCCATGTTTGGAGGAAGCCCCTCTGGAGACTCACTTGCCAATAAGCCCCAATACCCCTTCTCCGCCCCAGCCTGGCCCCCTGCCCTCAGCTTGTCTCCACCCACTCTGGTCATGGTGGGGTCCTTGCCCTCCTGGGACTGACTGGTTGTGTGGGAGGATGCAGTCTTCCAGAAGCCCCTTTCTTATTGCCCTGGACATCAACCAACTTGGGTTCTTTCCTCATGAGTTTTATAGACAGAAGAAAGCTAAACCAAGAAAAGGGAAGCAACAACAAATCCAGACTCTTTAGTCTTTTCCACCCTGGTGCCTGAGCCAGGGATGCCTCTGCCTGCATCCCAGAAACTCTGTTCTTCCAGGCTGAGCCCACCATCCCTGCAGAGCCACAGATGCCCCTCACCCTCAGACAGGAAGTTTACCCCTCTGAGCACTTTTACATTTCTCATATCATTTTGTCTTCACAACCGCTCTTCATCTGCTCTCAAGTGAAGAACTGGAAGCAGCAAGATGTGCCCAGGATTCCAGAGTCAGGAACAGAGCCCAGGGCTTTGGCACATGAGGGTGGTTGCAGGAAGGGCTGGTGCCTTCTCTCTGTTGTCCCTAAATGTCCCTCTGCTCAGCGGGGGGCTCCTCCTTTATGGTGGAAAATATGCTTCCAAGCCTCGTCCACTGAGGCGGGTCACTCCAGGACATCCCCAAACTTTCCCTCAACTGCTCTCAGAAAGTCCATTCCCCCAAAAGGGACAGCATCCCGGGTGGGCAGGAAGAGGCCCAGGCCGTGGCTGTCTTGCCAGCCAGCTGCTAACCCGGTAGGCCAATGTGGGGCTCAGCTTAGAGATTGATCAATCTTCCCTTCCATTCCTTCTCTTCAGGTGCTTAACCTGTTCATCGCCCTGCTATTGAACTCTTTCAGTGCTGACAACCTCACAGCCCCGGAGGACGATGGGGAGGTGAACAACCTGCAGGTGGCCCTGGCACGGATCCAGGTCTTTGGCCATCGTACCAAACAGGCTCTTTGCAGCTTCTTCAGCAGGTCCTGCCCATTCCCCCAGCCCAAGGCAGAGCCTGAGCTGGTGGTGAAACTCCCACTCTCCAGCTCCAAGGCTGAGAACCACATTGCTGCCAACACTGCCAGGGGGAGCTCTGGAGGGCTCCAAGCTCCCAGAGGCCCCAGGGATGAGCACAGTGACTTCATCGCTAATCCGACTGTGTGGGTCTCTGTGCCCATTGCTGAGGGTGAATCTGATCTTGATGACTTGGAGGATGATGGTGGGGAAGATGCTCAGAGCTTCCAGCAGGAAGTGATCCCCAAAGGACAGGTAAGAGTTATCCCCAAGGGACAGCCACAGGCAGTGGGGAGGGGCTTCAGGGCTTGAGTGACAAAGGGCAAAAGGGAAATGCAGAGGAGACCTTGACATAAAGAAGTTTAAACCATGCCAAGCTTTCCAGAGGAGTTGGCGTTTGAGCAGGGCCTGGAAGAAAAAGTCAGAATTCCCCAGGCAGACAAACAGGGAGTGGGGGGCAGCAGGTGAGCAAAAGGCCACCTTGAGGTGCTTGGGGATTGTCTTTCACACAGCTGTCCCATGCGCTCTGCCCAACCCAGGCTCTGTGCATGAGATGCTAATGAGATTTCTCTAAGAACAGGATGTTGTCCAAGAGCAAGGCTCATCTAATCTTTACCAGCATCCTAAGAAGTCCTGATTCTTATGTGACTCATTTAGCCAGCTTTTCCCCACAAGTGGTATCACAGGGAAATGACACGCTCCTTTTGGCACCAACTAATTTAAAGCACATTTGAGCCATTTGATAATATATATTTTTAAATACTTGGCACTATTCCTTACTACCTTAAGCTGGTATATCAACCCTCGCTAACCTCTTCTCAGTAACTCAGGGCATTCACCTCTGCCCAACGTGGGAAGGCAGGGAACCCTGTGAGGCCTCAGGAACATGCAGGGCTGCTGGCCAGGTTACTAAGTGGTCCAGAAAAGCTGTGCTTTGAGTCTGTAGCTCTCCCATAGCCTTCTTCCCTGCAGTCGTCACTTGTGTTCCAGCAGCTGCTGTTATCAGCTGCCATATTTTACATGCCTTTGTCTAGGTAACAGCAGAGAGCATGAACTCTGTGACAGCTGTCTGCCACTCAGAAGCATTTTCAGTTACTTTGGGGCTCTTAGGGAGCTCCAGGTGGCCAAGTTCCCTACAATTTTGACCTCTGGACAAATAACAATGGTCATGGCTAAAATCTATTGAGCCAAGCACTTCGTGCAAAGTGTTTTACAAGCGCTAACATATTTAGTCATCAAAACAGCTGAATGATGCAGATCTAGCCTTTTCAATTTTGAAGCTGGGAAAAATGAAGATTAGAGAGGTTAAACAACTTGCCTAAGGTCACACATCTTATAATTAATGGAGCCCAAGGATAAAACATAGGTCTATCTAGCTATAGTCTTCATGCTCACTTCACACACGTGTATGTATATATGTATGTACATGAGTTGCATATATGAAACTCTTGGCTCTGGCAGCCTGTGATGTGCAAGATCCCTTCAAATTTAGAACTGGTCATCTAAGGGTGGGGCCAGGCACCCAGTTGGGACCCTCTCTCACTAGCAGGAGCAGCTGCAGCAAGTCGAGAGGTGTGGGGACCACCTGACACCCAGGAGCCCAGGCACTGGAACATCTTCTGAGGACCTGGCTCCATCCCTGGGTGAGACGTGGAAAGATGAGTCTGTTCCTCAGGTCCCTGCTGAGGTAGGTATGTCAGCTTCCTGGAGAGGTTGGACAGCCAGCCAGACACTGAGCAGTGGGCTGGTAGAGGCTGGAGGGTGGCGGTGAAGGTGGGGTATTCCAGACTGCACTCAATCACATAACCTCATTTCTTATCCTGGGAATTTTTAAAGTCCCTCCCAACCTCCGGCTAATCCGCACAATTTCAACTTTCACCATATACCATTGCCACAATCTCACTCCAGGGTGGTTCTAGGTATAGAAAGAAAGAGTGAGACTCCACAAGCTGTGGGACCTAAGATCAGTTCCCAAACCTCTCTGAGCCGCCATTTATTCATCTATGAAACTGAATGGTAATGCCTACTTGGTAGGATTATTTTGAAGCTTAAATGAGATGCTGCTTGTCAAATGTTTAGGTCCTGCCTGAGACAAAGTAAGTGCCCAGGAAATGACAGCCAAGAAAAAAGGAAACGAAAGACAACGCACAAAGAAAGTCAAATATCTTTCAGAGCCAGCAAATAAGAGTTGGAGGTGCTCACAGATGAAGAAGTTTTCCCTTGTGATTCTCCCCCACTTTCTTTTGTCCACATCATTTTCTCTAGGCAAAAGTAGCCTGGGGCTAGGGAGAGGCTCTCTATAGGTGAGTAGTGAAGTGACAGCCTCACAGAGACTGAGCTGGAAGATTAGAGGTTTAGGATTGTCTTATACAAGGTAAAAATAAATGTGGTTTCACTTACAAATCTTGAAGGAAACAAGCTATAGGGAAAGAGGCATCTATAAAAGTTTAGCATCTTTAATAACACTGCCTTCAATTCAAGAATTTAATTTGACACCTAGCCACTGAGTGCTGACTTTGTGACAGGCACTGGGCTCAACAGAGATAAGGAAGTCATAATTCATCCCCAAAGTTATCAAGCCCTTCAGCTTGAAGAATGTCAGCTACTGGCTGTGTGCCCTAGGCAAGGCTGACCACAGCAGGGTAAGATAAGAGGGAGGGCTGGGACGGGATTTGGGGGCTGTGGGTGAACAGAAGCCGCTTGGTTCCCACTGGGTGAGGGCTGTCACATTCTGCACGTGGATGTCATGCTTCTTCCCAAATGTTCCAAGGACTCCCCTGGCTAGTTGTCCCTACTCAATGTTTTCAGTACACAACTTCTGGGTGTTGGTTTTTTGTTTTTGTTTTCTTGTCCATGACCCCGGCTAGGCAGGAGGGGCCTGACAGCGACAAATGGGGACCCGCCCCACCATTTGGGTCCATTAGCAAAGCTATCAGGTTTACTAATATGAGAGCAACTTCAACCCCTAATTGAATTCATCTGGAGACATTATAGGGCTGGAGCTGCCAGGAGGGCAGCAGGCCCCTGCAGCATTACTCCATGATTAAATATTCAAGCCCAGTGAATGCTGAGAGTCGTTATGGGTGTAATTACGGTGTCTCGGGCCATTGCCAGCACTCCATCAGGGGCCCCGAAGGTTTACAGTCTCACACAGCTAAGCCTCTGGGGCTCCAGGGAGAAGAGCAGTGTTCCAGGCCTCAGCTGGCGAGGCACCAAACATGGGTGCAGTTGAACCTGCAGTGACCGTGTGCAATTCCCCGGGCCCCGCGGAGAGACGAGTTCACTGCCCTGGGCTTCTCCCCTGCAGGGAGTGGACGACACAAGCTCCTCTGAGGGCAGCACGGTGGACTGCCTAGATCCTGAGGAAATCCTGAGGAAGATCCCTGAGCTGGCAGATGACCTGGAAGAACCAGATGACTGCTTCACAGAAGGTGAAGGGACAGCCACAGGCCCCCTCACACTGATGTTAATTAGGGCCAGAATCCAGTTGAGCGAACACTCACCTGGGATCCACAGAAGACAAACAAGCTGTGCCCACTTGCTGCCTCACTCCCAGGCTTGAGGGCGCCCGCATCAGACACACATGCCCCAGGTGGCAGCAGCCTGGCCGTGGTATCTGCATCTCATGCCCTATCAGACACTACGGAGAGACGTGGGCCTTCCTCTTTACCCTCAAAAACCCCTCCGTGAGTTCTAAACCAGCCCTCTTGAGTTGAAAGGGGGAAACGTAGTCACCACCATAAACACCACTGAAAACTCTTGATTTGGACCATGAAGGACCCAGAGGAAGGCCATGATGTATCTGCTTCCCTCAATCTTTTGTTTAGCTCTCAAAAGCAGAACCTGTGTCTTAGTCATCTTTCCTCTCAGTGTTTAGAACAGTGAGGCACATTACATTCTCAATAAATATTTGTGCAATGAATGAATCAAGAGTCCATTCTATAAAGGGAATTGTGGCCTCAATGCTTTCTAAAAATACAAAGAAAAATTTTTTAAATACTTGAGTTTTTATTAATAGTTTTATTTGACCCTGGGCTGGGCTGAGCTGGGCTGGACCCAATCCCCATCCCAGCCACGTCGTGGCCCTCAGTTCAGCTTGGGCTTGGAGCATATCCCCCTCAGGCACCCCCAGGCTCATACGCCGTAGCTTGTCTGTTTTCCAGGTATAGGTGGGCAGAAGGACAGGGACCACTGGTGTCATCTGTAAGAAGGATATGGCTAGGAAAAACATCAGAGTGGGCAAGACTGATGATGGAGAGGAACACCTGTTGGTCTGGATTGAGGGACTCCCTGGAAGGGACCCCTCTTCACCCATATTCCAGGAAAGAATTTTTTTCCAAGGGCATGACAAGTGGGTTTCCTAGCAGAATGGTTTCTCTGCAGCAGGAGCAGAAATTATCCCCTTTGCCCACTGGCCATCACCCACCATCAGTCACCTCTCCCCACAGGATGCATTCGCCACTGTCCCTGCTGCAAACTGGATACCACCAAGAGTCCATGGGATGTGGGCTGGCAGGTGCGCAAGACTTGCTACCGTATCGTGGAGCACAGCTGGTTTGAGAGCTTCATCATCTTCATGATCCTGCTCAGCAGTGGATCTCTGGTAAGGGGAGGCATAGTCCCTGCCCCCAAATCCTCCAGATAGGAATCTCCTACAATCCAAAGGCCCTGGAGCTGCATCAGTGGGTTCTCCTGACTACTGAGCTGAAAGTCCCCAACCTTGTCACTAGAAGGAGAGCTGCAGCCCAGGGCGAGGGCCCCTCTTAGCAGCAGAGCATGGGAGGTGTTCAAGGTGCCCGTGCCATGGCCTGCCAAACCTGGCTGCAAGCATTTGAGTGAGTGAGTGTATGCCTCCCACTATATAGATGGGGAAACTGACACTCAAAATGTTTAAAAGGCTTGTCCAAGGTCACCCAACTTGTCAGTAGCAAAACCCAGCTTTGCCCTTATTTAATCCCGAAGCCCCTGGCTTCCCACCCTCTGACCTCCCCAACACACGGCCTTCTCTGAAGGCCTACAATACAAAAAGGACTATCTTTCCTTATCAAACCACTTCCAAATGCTACTAATTACTCCTTTAAAGCTCACAGAAAATCATTACAATAGCACTTCTCAAAATTTAATCTCCCAGGAGCTTGTTCAAATGCAGATTCTCATTCCATAAATCTGTGGCAGGGCCTGAGAATTTGCATTTCTAACAAGCTCCCAGGTGATGCTTGCACTGCCCTCAGTGACCCACACTTTGAATAACAAGGCCTTATGGGAATAGAGATTCAGAGTTGGGAGACATCTCTAAGGCCCTGGCTGAATACTGGGCTTTGTAGAGCAAAGAAGGAAGGTTCTCTCTGTACTGTGGCTTAAGCTCCTTACACAATCTCAACTACTCCGCCAATAGACCAAGGTAGACATAGACACAGGAAGGAACTTAACATCCTCAGTTGTTTGCATTTTCTAAATGAGGAAGTCAAAGCCCAAAGGCATGAGGGGCTTCAGGTAATTAGTTGAAGGGCTGGGAATAGAGAGTTCAGGGTGGCAGCTAATTAGTTGAAGGGCTAGGACTACAGTCCCGCTCTTCCGATTCACGGCTGCAGTGTTTTCACTCATCTTGCGCCTCATGTGTCATTTACTCTCTAAAAACAACCTACTAAATATCTAGGCCTCCATCTAAATGCCAGATTGTCCAGGAAGGGTGACAAAACTGCTCGGTGCATGGCCTCAGCTTTAGGGAGGAGCACAGGTGCGGGACGAGGCAGGATCTGAGAGGTGGGAGGAAAACTAGGGTGGCAATGACGCAAAGCCAAGGGAGAAGAGAGTTTCGGGGCAGGGACTGGTCTGCCATCCTGGGTGGAGGGGGAACTGTGTGACAGCGGCTGCAGGTGCCCAGTGCCCTGGCACAGGGCAGTGATTGTCCACACGGACCTCAGCACAGTGTAGAGGCCAAATTGCAGGGGCAAGAGAGGGCTGGGCAGTGCTTTCTCCACACCTATGCTCTTCTCCTTCCAGTCCTGCTGGATTAAAGTAATAACCACATGTGCTGTTGGCCAGGTGCTCTCCTCACTACGTCATTGGCTTGTGGGCCTTCTCTCCTCATTTTGCTTCTCTGCTCTCCCTTGCTCCCTCCCTTCTCTCTCTAGCCCCCTTCTCTTCACACCCCGGTCCACCCCCCACATTCCCAGGAGGAAGCTCAGACCTGCAGCACTGCGCCAGCCCAGCCCGCCCCCTGCAGCTCTCCCTTCCCTCCCATCTCCTTCCCCTGGGACTGAACAGGCTGGCACCAAATTTAGCCTTCTTTAGGCTTTCACTTTGAGTACCTTTTAAAAATAACATGCATTTTCATCTGACAAAGTAAAGTACCAAAAAAATACTTGGAAATTATAGAAAAAGGTAAAGAAAAAAAAATCATCCATTTCCCTATCACTCAAAGAGAAACTCACTTGAAAACATGCTGGTATACCTCTTTCTCATCTTGCTTATGCATAAAATATACACATATTACAAAAATAAAACCACATTTATGCATTCTATAACCTCAGTACATTTTTCTCAGTAAATAGTCTCATATTATCTCATTTAATGGCTATAACATATTCCTTTATAATTTACTCAACCAATCTCTATGTTTACTTCTAATACACATTGAATATTCCCTGTGTGTACTCCCTTAGAGACGTTGGATAATCTCCCATCTTAGTCCTCGTCCTAGACACAGAATCGGAAGCTGAGAAAAGATGTGGGTTCAGTTGAAATCCGGCTTCAGCTGATCCCATGGAAAGCCCTGAAGCACAAAGGACACCAAAGAGCTGTCCCACCTTGAGGCAAAGAAGACAGCCTTGTAAATCCCCCTGTCACTGTCCCTGGCTGAGGGCTGCCCCAGCGGGGAGGATAAGCATTTCCCAAGGTGGACACAGTTTCCTCCAGCCCAGCGAAGCCATCAGGAAGGGGGTGTCTGCATCCATGAACTATTAGCAGCCGACACTCCCAGCAGATGAAGGATAGGGGCACTGGCCCCTTAAAGAGGACCTGAGTGGGCACCAACAGCCTCCAGTGTCCTTAGGATTAGTGCTTGAAGGAGGAATTGCTAGGTTCCTGAAACCTCTGAGTCACATTGCCAATTTGGCTTCACACAGGACTGGCCAGGTTTTCACTCCTGCCAGTCACTTGTAAGAGTGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCATGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCTGGGAGGCAGAGCTTGCAGTGAGCCGAGATCCCGCTACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAGTGGCAGCCCCCCACAGCCACAGCATCCTAGGGTTTCCCCTATTCTTTTCCATCTCCACCAGTCTGATAAAAGAAAAACAGCATCTTCTTTTAATTTGAATTTCCTTGATTACCAGTGAGATAGAACATTCTTTTTCAGAGGTGTGTTGGCGATTCTTACTTTCATTTTTGTAAACTGCCTGTGAACATCCGTTGATGCTTTTATTTTTAAACAAAGTCACTGTTAGCATCCCTTTGGTTCACTTCCAAAATCCAATTTGAGAAGGTTTTCTTTCTCCCTTGCATTCAGCATCCCTGCAAGTATCAGCAGGGCTGTTTGTCCCCATTGGCCCTGAAGAAGTCATGCTTTGGATCTCACAGTACCTCCTCCACTATCCCACTTTACCTCCCCACAGCAAACAAAATGCTACTGGCAAGCTGACTACCAAATCCCCTTCCAGGGACTGTGGTCAGGGCTGGCTGGGTCCTTCCCTATCAGTCTGGGCCCCAGTGCTGTGGGTCCAGGCAGCCTGCCATTCTGCATCCTTTCTTCCTTCCAGGCCTTTGAAGACTATTACCTGGACCAGAAGCCCACGGTGAAAGCTTTGCTGGAGTACACTGACAGGGTCTTCACCTTTATCTTTGTGTTCGAGATGCTGCTTAAGTGGGTGGCCTATGGCTTCAAAAAGTACTTCACCAATGCCTGGTGCTGGCTGGACTTCCTCATTGTGAATGTGAGTGGCTCAGTGGGAACACGTGGGGTTTGGGGTCCTCCCCTCTGACTCCTGGCTACAGCTAGGGCAAAGGAAGCTCAGAGACCTGGCCCTCAAGGGCTACTCCAAAGAAAAGTTTTGAGCATTTAGTGAGGAGTCTGAGAATAGCCCTGACACCCATGAGTGGTAAAGTCCAGGCAGTATGTGACTGTGACACAATAGTGACTGTTAGGTGAGTTATTAGGTCTCCATTCAGAAAGATCCCTGCAATCCTCTGCATTTTTACATCCAAAGTGAGATGACACATGCAGCTCTCAGAGGTCTTATGGCCCTGCATCCATAGATGGGTCTTCTGGACCTTGGGGAGCCATGAACTGTGAGCACAGAGAATCTTCCCACCTGCGGTTCTGTGGTCCTCACACCTGGCTGCACTTGAGAATCACCTGGGGAGCTATGAAATATGCAGTGACCGACCACCACTACCCCAGAGATTCTGATCTTATTGGTCTTGGGTGGTGCCCAGGCACCTGTATTTTATAAATGCTCCCAGGCATTTCCAAGGTGCAGCCATGGCTTAGAACCACTGAGCTTATACTGCTGCCTCCTCCCCAGTGGCATTGAAAAGAACAGAGGGCGCATTTGGAGAGGTAATGAGCTGGAGTTCATGTGTTCACACTCATGGTAACCTGTGGCCAGTAATCCCTGCATTAGAGAATTGACTGTGTGCTTCTCACACATCAGACATGTGATTCCTCAGTCATCTCTTCCTCATCTTAACCACCACATTGCACTATCACTGTCTAGGCCACTACCTTCTTTGCTTTGCAGCATGGTTAACTGAGTTATCTGTTCACTGCCGCATAGCTGAGGGTTTCTGCAAGCAGGGTGAGGCATGTGCTGTGTTGGTCTCTGCCTGCCCCACTCAAGCAGCTGCCAGCAGCAAGGTCTGCCCAGCAGAGGTGTCAGGAAATGTGTCTGTATTTGGTGCTTGCCTCCTGAAGCTGGATGAGGTTCAGCACAGAACATTCTGTCCAGCCTCCTGTGCTGGCAGGTCCTCCATAGCAGGCCTGTGCATTCAATTCTCTTCTTCCCTGATGCCAATGCACTAGCAGCTTCAGGCCACTGTACCACAAGGATTAGCTGTGAGGCCTGAAATCTGAGTCATGGGTGATAATTCTGTGCATATGAAATAATTATTCATAAATGAAAGGAAAATTATTTATTCCGGTGTCCCACCCCTTTGGGGGCACAGTCACTCCTGCTTATGTTGATCTCACCAGTCTCAGTCCTAATCACCTTAAACGGTCATGCACTCATTTCATCCATCTTCCTACTGATTCTTCCTTCCAACCATCCATCCTTCCTTCCATACATTTCACCTTTTGTATCCAACCATTCAGTCTTCCTTTTTCCTTCCTCCCAAACATCCATCCATCTTTATTTCTTCCAGCTGTACATCTTCATCAGTCCTCCCCTTCCATCCATCCATTCCCATTCTTCTTTCCCTCTTCTCTTCTGCCCAATCTTTTATTCTAAACTTCCTTCCATCTATTCATCATCCTTAACCCTTATATCTTTCTATCTTTTGATTCATTCTTCTATCCAACTGTCCAATCATCTAACATTCTGTTCACTCATTCACGCATCTTCCATCCAAGCATGCTTCTACTCACTCATCCTTCTATCCACTCATTCTTCTATCTTTCCATTCTTGTTTCAACCTATCCATCTATTCTTCCTTTCTTCCACTTATCTGTTCATTCATCCCTTTCCCTTCCATTCATCATCCATTCATCCATCCTATCAACCTTCCAGTCATTCACTAATTTATTCCATTCATCTATTCTGTCCAGCTCCCCATCTTTCCATTCATGCATTAGTCATTTTACCCAGCCATCCATCCTTCTCTTATCCACCTATCTCATCTATCCATTCACTATTCCCTGCTTTCATACTTTTCCTTTCTTGTTTGTTTCTCCCATCCTTCCATTCAACTATCTATCCTATCCATCTTTTTGTGTCTGTTATCTTTTAAAATTTTAAAAAATATATTTTATTGTATATATTTGTATTAGTCTATTTTCATGCTGCTGATAAAGACATACCTAAGACTGGGCAATTTATAAAAGAAAGAGGTTTAATTGGACTTACAGCTTCACATGGCTGGGGAAGCCTTACAATCATGGCAAAAGGAAAGGAGGTGCGAGTCACGTCTTATGTGGATGGCAGCAGGCAAAGAGAGAGGTTGGGCAGGGAAACTCCCCCTTATAATACCGTCACATCTCATGAAACTTATTCACTATCATGAGAACAGCATGGGAAAGACCTGCTCCCATGATTCACTTACCTCCCACAGGGTCCCTCCCCTCCCACAACACATGGGAATTCAAGATAAGATTTGGGTGGGGACACAGCCAAACCATATCAATTTTTAAGGGATACAAAATGATGTTATAAGATATATATATATATAAATGATTACTATAGTGGAACATATTAACATATCCATCATCTCACATAGTTACCCATTTCTCCCCCTGTGGCAAGAGCAGCTATAATTACTCAGCGAGCAAAAATACTGAAAACAATATGCTATTATTAACTATAGTATTCATGTTATACATTAAATCTATCCTATTTATCATTGTATCCTTTAATTCATCCCTCTATGCAATCACCCTTCAACTTTTCATCCAGCTGTACTTCTTTCTTTCTTTCCTTCTAGCCATCCCAATGATCCTATTTTTCTTCCATTTGCTATACATCTAACTATTGAATACAGGATTGTCTTATGAAGTGCTTTGTAAGAAACAGAAGCACAGAATAAATACTCACTATCTCCAGGATCTTACACTTTAGTCTTTCAGCAGACAAGACACACATGGAAATAAATGTATTATAAGGGTGAATGTGTTGAGCACTGCTGGGGGCAATTAGTATCTTGGAGACACAGAGACATGAGCCTTCTTCCACTCTTGGGGTGGGGTGGTTGAGAATTCTTCCTATGGGAGGTGGCATTAGATGTGGGGCATAGAAATGAGCAGGACCAAGGAGCTGCGAGAGGACTGGGGAAGGCCCGTCTTAGCTAAAGGCTGACCTTGAACAAAGGCACAGACACAAGTATGCTCAGGGAACAATTTGATTTGGGTAGAATGTGTGGGTGAAATATGACTGGAAAAGTAGGTTGTGACCATATGACACAGGCTACAGAGTATGGAGTTTATCCTGAGGGTGATAAAGTACCAGTGATGTTTGGGAGTAAGAGAAAGACATGATCATAATCAGATTTGCAGTGGAGAATAGATGCATGGTGCAGTACTAGGGCCTGGAGACTACTTGGGAGGCTAGTGGGCTGAGCAGTGGGAATGGAGGTGGGCTTGGGTGGACAGACTTTGCTGGAGAGGCACAGCACCTGTCTGCCCCAGCTGCAGATTATCCATGTGAGGCAGACTTCCCAGGGGGCTGAGCAAAAGACTGAATAGGGTTCAGCATGGAACTGGCCCCAAACCTCCCTACCCTCATGCACCCAGGCAGCTGGGTCTCACCTGCTGATGTCTGGAGCCTTGCATGAATGAAGGAAGAGGGTCCCTTCCATTCCGGTTCTTTGTCCTGGTGCAATAAGCCACTCATCCAAAGAATGGACATTGCTTGGGGCTCTGCCACTCAAGTATAAAGCCCTTCTTTTCTGAAGCTCCTGTGTGCATCCCACCTAACTGCCAGGATAAGCTGAGCCTGAATTCAGCCCACCATCTGTGGTGTGGCTAAGATCTCACAGGACAGGGTCATAGCCCGTATCTATGACATCTCCCTGGCACCTTCAAAGTTCCTAGCAGGGGAACTTTTTTCATGTAAGTTTCTGTCCTTTGCAGTCTTTGGACCACCACTTAAAGAAAACTGACAGACCAAAAGGGGCTCACAAGTCAGTCATTATATCTCAGATATCTATTGAAGCTAGATCTCATGGACCCATACCAGAAACTTTTTGAAGAACTGCAGAGCTATCAGAACTTTGGAATTCTCTTAGTCTGACTTTTTCATTTTACAAAGGGTGAAAGTACCCTGGACTAGCCAGGCACTAACCCAAGCTCACAAAAAATCAGGGTTCAAATGGGAATTAGACCCCACCCAAACCACTATTCTTTCCACCCCACTTCTCTGAGCAAATATAATTCAGATTACTATATTCCTCAGAATAGACTCTCAGCATAGCCCCTAGGAGGGAACATCATGTCTCCAAGTCTCTGAAAATCAGTTTCAAGGTTGTTGAGGCCTGACTGCTGCCCCTAGAAGGAAAAGGAAGAGTTGATTTAATACACTGGAAGCTTGGCCACCTACGTACCAGAGGTTGGTGATGTTAGAGACTGAGTTTTTCTTGCCCAGATTGTTTAAGAGAGTGGTGAATAAAATTTTTAATAAAAGGGAAGAGACAAAACTCTTTCTAGACCTCTCATTGGAGTTCCGAATAAGGTGGGAGCTAGGATGATGGCTTAGACGTGTAGGAATGGGAGGCTGGGCAGGGAGTTCCTGTCTCCTTCCACGAGGACTGCCTTTCTGGAAACCTAGAGTGATGTTTCTGCCCTCTCCTGCACTCAGATCTCACTGATAAGTCTCACAGCGAAGATTCTGGAATATTCTGAAGTGGCTCCCATCAAAGCCCTTCGAACCCTTCGCGCTCTGCGGCCACTGCGGGCTCTTTCTCGATTTGAAGGCATGCGGGTAAGTCTGATCTCAAAACTTCTCTCATCTGGCCAGGCACGGTGGTTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCACCTGAGGTTAGGAGTTCAAAACCACCCTGGCCAACATGCCGAAACCCTGTCTCTGCTAAAAGTACAAAAATCACTACCACCACGAGGTGTGGTGGTAGTGAGCCGAGATTGTGCCATTATACTCCAGCCTGGGTGACAGAGCGAGACTCCGCCTCAAAAAACAAACAAAAAACTTCTCTGGTCTGCAGGTTTTGGTAACCCTTCCCCCTTTATGCCCTCCATCCCACCAACCCTGAGCTCCGGGGCCCCACCTCCAAAGGTAGGCTCTGAGTCTTCACTTCCTGCTCTGACTTTGGTGGTGCTGCATGTCCTAACTCTCTTAGTTCTCTTTCAAGTCTTTTTGTGGGCCCATCTTCCCTTGTCACCCATAAATATTACAGCCACCAGGACCCAGACCTCACCCCCTTTGCTCTTAGTGAAGTAATCCTGAATCATCGCTCCACAAGCATCTATTATCTCAATCACCCAACTCTGCCTCTTATTGCCAACATCTCCAGAACATCCCAAGTTCTCTGTCTTTCCATGACAAATGAGTCTTTCCTCTAATACTCCATGAGTGACATCATCAGCTCATTACCTCAGCTGCCACCCACTGGAATGCTTCTTCTCTCCTACTTAATGCAATCATCAGGTCCTGTAAATTTTTCCCTTCCATCCACCCCTTTCCTTATCCACCACTGTCAGCCTCCACCTTGCCCTAACTGCCTCCGTCAGGACAGAATGCTCACAGCCAGAGCCTTGTCTTAGAGATTGCTGATTGACACCCACCTCTTCCTCATAATTACAAATGGCTCCCCAGCAGAAAGTAAGGTTTTGCTTTGCTCCACTATCATTGGCTGGAAGTAGTCATCATAGCTTCAAAAACAAAAGTGGGCTTTTGAAATTAACTTGTACCTTTACAAACACTAGGACCCGAAGATTGAAATAGTTTGAAATTTATTCTATTTTGTATCTAATAGATTCTCATTTGCTTATTTACAAATAAGCTATTTTGTGAATATGGATTTAGGTTTTCACTGGTTCTTGACCACGGCATACTTTAAATTGTATTTAAATTGTGTTTGTGTTTTATGTATGGTATTTAAGGAGAATAAGTCAAGGTTCTAGAAGTCTGTATGTGCAAGTATAATACACACACTCACACTTCTAAATGAGTGCAGTATGCTTGTAAACTCATCCATCTTCTCACTGCAACCCACCCTGAATGAGGTTAAGGGAAGAAGGTCTAAATCCATCTTCAGCTGCCCAATGTCTCTCAGTCTTCTGAGTCTGAATTATTGCACTAATTAACCCACTTCTCTTGCTTCTTCCAAAGGCCACTGGCTCATGAAGCAGCCTGAATGAGTATAGAAAGAGATAGAATCCAGCAGAGGGGGTGGGGCATTGGGAGGTTCCAGGTCTTTCTCTATCTCCCACCAGGTGGTGGTGGATGCCCTGGTGGGCGCCATCCCATCCATCATGAATGTCCTCCTCGTCTGCCTCATCTTCTGGCTCATCTTCAGCATCATGGGTGTGAACCTCTTCGCAGGGAAGTTTTGGAGGTGCATCAACTATACCGATGGAGAGTTTTCCCTTGTACCTTTGTCGATTGTGAATAACAAGTCTGACTGCAAGATTCAAAACTCCACTGGCAGCTTCTTCTGGGTCAATGTGAAAGTCAACTTTGATAATGTTGCAATGGGTTACCTTGCACTTCTGCAGGTGGTGAGTCAACCATGAGATCAACCATATCTCTTGCTTTGGCCGCTCAATAAAATGGAGAGTCTAGGTTCCCAGAAGATGCTATGTGGGGACTTTGCAAGGAGTAGCACTCAGTGTGAACTGAGGTCATTCTCTATTCCTACAGACTACACCATGAGGCTAGAGGGCCTTCCTAGTGAATATGATATCACAGTGGTGCAATTACACAAGTGTGGGCTAGTAGAATAGCATAAAGGGATGGGCCTGCCATGGGATTATCAGGTGGTGAGTTAAAACACAGAACCTGTACAAAATAGACACGGCATAGCCACATGTTCAGTCTTCAACTTCACATCATTTCCTAAATGGTAATAAATTATGTCTCATTAGGTCTATTGTAATGATATATAATGTTGGTGATAATGACAGTCAAACACATTCATCCTGTCTTTCTTTCTTTCATCATCCAGTCAATCACTCATTTCTTGCCTTCATCTACCTGTTCATTCAGTCCTCTGTTCATTCAAGTGTTAATACAATTACTTATACATTTGTCATGCAGTTACTCTTGAGTGTCTACATTCCACCATTGGTTCTTGCATTTATTCAACAAATAATCTCTGAGTAGCTCTGGTATGCCAGGCATGGTACTACTCAGCCCTGGGAGATACAGTCAGAACTGGGCTTGGACACTGATGCTGTATGAGAAACAGACTAATAGGTGAATTAAAACAGCTCATTAAGTGCTGTAACAGGCCTAGGAGTGTAAGAACTAAGATGTCTTGCATGCTCTGAACATGCTGGAAGGCACACCCAGTTTCTAGACCTCAAGAATGCTAAAGCTGAAGGCAGGAACATGGAGGTCTTCTATTCTCAACTTCTCCATCTGGAAGATATACCATTCTTACATCATGTGCATAAGGTGGGAAAGTGGCTGAGAAAATGGCAGCCCTGAGAGGGGAAACCTTAGGCAAGTGCACAGAGCAGACTTGTAGCAATTGGCACTAGAACCAGACACCTAGACACTCAGTCCTCAGTCCATACTACATTCTGAAAGGCTCTGTGTCTTTAATGCTACTCTATTCTTAAACAGCCACATGTGGGATTACTTTAAATCATCATATGAGATTAGTCAAGTAACCCAAGGGAACTTTTCCTTTCATTCTTTCTGCACAAGCCCTTTCCAACACAGGACTCTCTCCTCTCTCACTGGGCCACCATGGCTTGGGAAGGGTCCAGTATAGATGGATGGGCAGACACTGAGTGGCCTCTGCTCCTTGTCTCCAGGCAACCTTTAAAGGCTGGATGGACATTATGTATGCAGCTGTTGATTCCCGGGAGGTGAGTCTCAGCCCACTGTCCCTACAGCCTTCCCCTCTGTGCATTCTACCCATATCAGTCTGCATTTGAAATGAAGTGACGATGACCCTGGTACTGGAAGAGGCGATCACCCAGCAACCTCACACTGTCCCTCCTTGCAGCAGTGTCCAGCTGGCTCTCTGTTGAGTGTTTCAGCCACCGAGGCTTTCCCCCTTTCATCTACACTCCCCAGGCTTCTCGAGGTTTTCCTCTGTTCACTGTTGAGGAAAGCATCAGATAACCTCTAAGAGACAGTTCATGTGAGCCACTCCCTGGACAAGGGTGGGAGCAAAGCAAGGGTAGTAGCTAAATCAAGGTGCATAGGGAGGAATGAGGTAGAATCTCAGGAACTATCATAAAACCCAGGATGGGCCAGGCGCAGTGGTTCATGCCTGTAATCCTAGCACTTTGGGAGGCCGAGGTGGGCAGATTGCCTGAGCTCAGGAGACCAGCCTGGGCAATACGGTGAAACCCCATCTCTACTAAAATATGAAAGAAATTAGCAGGGCATGGCGGCTTGTGCCTGTAGTCCCAGCTGCTCGGGAGACTGAGACAGGAGAATCTCTTGAACCCGGGAGGCAGAAGTTGCAGTGAGCTGAGATTGCGTCACTGCACTCCAGCTTGGGCGAGACAGCGAGACTCAATCTCAAAAAAAAAGAAAAGAAAAGAAAAAGCCAGGCTGGGGAGACACTATTGGGGAATGACCACTTCCTGCTGTGGCAGGCACTGTCAATTCCCTGCCACCTCTTCCCTTAGCCCACTTCTGATTTCACCTGCAGGCTGCACTTCTGATTCTCTGTATAAAGACTCCAGGGCTTAGGAGCTTGTGCAGCAACTTCTCCCCCAAGTACCAGGGATTTAATGTCCCTAGGGGCAACCCTTACCAATAAGTTTTGGGAGTCTGAGGATAAATACTCCAGTTTCCCTGTCCCTTGGTGATATAATCCTGCATGTGCTCTACACAGTTACTCAGAGGACCCCAGTAGGATTGCGCTTCAGTTGCCCACAATGATGCCCTGCTTAACAACATCCTCTTCACAGGCTCTTCTCCCATTCCCGCTTATTCTTCCCCACTCCCTCATTCCTGCTTCCTTGGATCACCTCCCAGATAAATCACCTGCACCCAGGTCCATGTCTTAGGCTTGGCTTTCAGAGTGAACCTGAACTAAGACACCCACCCTCCTTCCCACTCTCCCTTGTCTGGTCACAGGTCAACATGCAACCCAAGTGGGAGGACAACGTGTACATGTATTTGTACTTTGTCATCTTCATCATTTTTGGAGGCTTCTTCACACTGAATCTCTTTGTTGGGGTCATAATTGACAACTTCAATCAACAGAAAAAAAAGATAAGTGGTGCTCAGGAGTTTCTGGTTTCTGCTGTAGTGGTAAGCCTTATATTTCCTGTTCCCTGCCCAGCTCTGGCCTCCTGACAAAAGCCAACACTACTCACAGCCCATCACTTTAATATCAGTGCTAACCCTTGCCCTGTGATTGCTGTTGCAGGAAGAGTTGCATTTCTCTCTGTCAGCAATGCCCCACTAACATCCTCACTCAGGATTCACATCTCAAAGGGATCTTGGTCTTCCCAACTGGAACCAGGTGTGTTAGATGTGTTCCAAGTGAGCCCTGGGAGAATACAAGCAGGGAAAGAAAGGGATCTAGAACTTGCTGACCTGTTGGGGGATATGATCCCCACAAAGGACTGAGTCAGGGCACCTCAGGAAAGGCCATGGCCCAGCACTGGCAGAATCCCAACCCTCCTTGGGATGGGGAGGAAGGCTTGTGCCTCCAAGGGTGCCGACTCTGATTAATAGGAATCGAATCCCAGATAACCTCTCATGTTTATAAGAAAATGTCAGGGCAATAAAGGTATAAAGTATTTTCCTATTATCCTTTCATTTAATGCCTTCCATACCCCTGAGGTAGATAGGCGCTGTTCTAGATTTGCGTAACTGAGATCCCGAGAGAAAAGGGGCTACATGGATAAGTCACAGAGTACATGCAAACCTGGGTTTGCATTCCTGACTCAACTACTTGTGGATGGCTCATGCTGGCAATATTTTAAACCCCTCTGAGTCTCAGTTTTCTCATTCATTCAATGGGGCTAATAATACCTACTCTCTGGATTGTGGTGAGGGGTAAACAAAATCCTGAACAAAGATCACTGAGCATAAGACAACTTTGGTGGTGCAGTTTCTGGCACTGTAACAGCAGAGGGGAGTGTGACTGGCAGCGCCTCAAGAGCACCAGATGCTGTTGCTATTAACAATTTAAAATATATACCTACTAGGGCAGGCATTCCACTTACAGGAAATCCTTGCCCAGGGTTCTGAGCATAAGAAGAGGACAGGATAGGTCTGGGACAAGACAGTGCTATTTAGTACCCTGGGGAGGTTTGAGATTATTGAAGCAATCATTTCTCTCATTTTACAGATGAGAAAACTGAGGTCAAGAAACAGATTACAGAGGGAGATATTAGGTAACGGAAGGAGATGAGCTAGTGACCAGGCTGGGGTCACTGAGAAACTGCATCTACTGAATCCTTGGTCAGAGTTCTCAGCATGACCTCACCTGGAGCTCACCTACCCACTGCCCTTGCCCTTTCCTTCTCTCCCCCCATAAAGATGGTCCTCCCAGGAAGCCCTTTGTGTCCACAGATCTTCTCAGATCTCCTTTCAAATTCCACAGATCTCAGCCCAGTTTCCCAGCATTTGCAGGGACTAACTTACATAAACATGTTATTAAAATAGGTGGCTTTCAAATTATGTCAGGAAGCTAAAGGTGAGAGTTTGAATGAAGAAAATCCAAAGGGCCCCTAAAGAAAAACTAATACCCAATCCTAAGGGAGGAAAACATCTTCGTTGGTGCAGTTTCTGGCACTGTAACAGCAGAGGGCAGTGTGACTGGCAGGGCCTCAAGAGAACCAGAGGTCCCTCTGAGACCAAGGACCCAAGGATAAGGCAGTAATTGTCCTCATCTCCTCCTCTCCAGAAAAGATTTGAGGGCTTCTGTCCCTCCCAGAGTCTTTGCTTCAAGGTCCTCAGGAAATCTCTTGCCCCCTGGGAGGGACTGTGACTCTAGAAGCCCAAAGAATTTTACTGGAATGAACAGGCTAGATCCAAGGCTCAGATTTGAACCAACATGTACCTTCTCCTTGAGTCCTGTTTACCAACCCTCCCCACCCTTTATCCAGCATCTCTGGATCCCTGGAGAACAGAAACTGGCTTTGCTTTAGAGACCCAGGCAGGGAGTCAGCAATGGAACTCAGTCCCACCGTTCCGACACTGGATGTGGCAAGACACTCCTTGATGCTGGGCTTGGTTTGTCTATCTGCAGAGGAGGGGAGAAGGTTGATATGATCCTTTCTCTCCTGCCTGTTCTGATTTTGGTATCCGTAGGGGGTTCCTCTTGCTTTAGTAACAGTGTGGCCCTCTCTGCACTTAGGGGGCCAGGACATCTTCATGACAGAGGAGCAGAAGAAATACTACAATGCCATGAAGAAGTTGGGCTCCAAGAAGCCCCAGAAGCCCATCCCACGGCCCCTGGTGAGCCCCAGAGGTTTCCTCTAGCACAGCCTGTCTGTGAATCCAACATGACAGTCTATGCCTGTGTCAGGCAGGGGCCTGAGGGAGTGCTGTTTATGGAAGAGAAGATCTGGGTTGACGTTGGGATGAGGGTGATGAGGGTTCCTAGGCCCAAGGAATGAGAAGTTCAAAAGAAGCAACTCTGGTCTTCTGGAGTAAACATACCTTTTACAGCTATTTGAAATGAATCTGTTGGGGGAAGCACTGAGTTTCTCCAACAGTTTTAGAGTTCAAGTACAGGATTGGAGGCAGGTTAAGCTAAGTGACAGTAAGGGTAGTTAGAACCCAGGGATTAAACTTGTGTGGTTATGTATCATGGTCTAGGTCGCAGTAGATCTCCAGTATCACCCTATATTCTACCCCACGCCTACAGGTATAGAGAGGAAATGACAACAACCCAACTCCCCTGTCAGGATCCAAGATGCAGTCATGGTAGTGGTCAAGTGGCCCAGTAGGAACCAAAAGTTATCCCACTCGAGTTTAAAGATTGGGCAAAAATTAGGGCCAGACTCTAGTTTCACTCCTTTTTTCATAAAGATCTCAAGAGACATGGCTTTGCGTTCCTCCATTAAATGTACTACCCATACCACACATGATATCACCTAAGTTCTTCTCAGTCTTCTGCCAGAAGATAGAAAACATCAAGCCTCTGAGAGGCCCTAAGATAGCACTTTTTTGGGAATGGATGAACAGGTTTGGGGGTGAAGATGTTGATGTGTCAGAAATTATTGTTGATCGTCTTCAAAGAATTTCTGAGCCATCCACTCCAGTGAATGGAATTGGCCCTGTGTCCTTCTACATAAACTTTCTGTGGCTAAGATGCAGCATTAGTGAAGAAAATCACAGGTATTGCTGATGACACAATTTCATTAGCCCTTCCCCACACAAGGGAAAAGTCCAAAGGACATTTAACAAAGGAACAAGCCTTTCCCTAAATGAATTATGGAGACATGCATTCTGTTAGCCAAGCTATTAGATTTTTTTAAATCAAGGTATAGGATTTGCATAATTTACTTCAAACTTTGTCAGAACAAAGTGAAAAACATTTACAAGAGAAAAAAGTGATGACTTTTGACACTGTATTTTGAAATAGTGGTCATGAATTTATTACTAGTCTAGATTCTGATAAGAAGTATTACAATGGGGAGTGGGGAGTGATAGGAGGTTCAGACCTCTGTAGGGGCACAGCTTTCTTCCTAAATCACCCATACAATCAAGATAACTGGATTTGAGTGAACCTGAGCAAAAGTCCTTAGGCTCATTGAGTATCCCTCCATCAACAAGTTCAGTGGAGAGGAGCAGGCTCAAAATTAAAATTAGGTGAAAGGCTGAGTCCCAACCACATTCAAGTCATGGGTAATGAGAGGAGGACCCTAAGACTTACTGAGAAGCAAGCAGCTGAGTCCACACTGGCCTTTACTTGTGGCTGGAGTAAGATTGAATCCACATTTGAGCCAGATGCCAAGGAGGTGTGTGCTAGGGACAACCCTTGGCAAAAACATGAAAGTTTTTGGCACCTTCCCCCACATACTACTCAATCTCTCTTTGACATCCCAGCTTCTTCCCTGTCATCACCCAACCCCATAGGCTGCCAAAGAAAGCTTCCTCCCCTCAAGGAAGACAACATTCTTAGGACAAGATAAATCATATTTCAAAAAGGGACCCTGGAGAGGGCATCTACCTACAAAGGCATGACCCTAATGTTGAAATTCCAGGCAGTAAATAGGAGTGGGAGACGGAGTTGATGGTCAGGCAGGAAGACCTGCAACAAAATCACCATTTCTCCTAATTAAGCTGCAACTAAGATCCTCCGAGGATGGCACAGGGGGAGACCTCCACATGTGGAGGGGAAGTGAAACCTCATAACCAATTCTGTTCCTGAGATTCTCCATCCTTTGAATGGAAGAAGAGTGGATTAGTAATTCACTCTCAACCTTTTGCTAGCCTTAGTGGAGGAACGTGGCTCTCCCCTTAATATTCTGGCCCAAGCAAGGTAAACACTCTTTCCTTTGTTCCCTAATTTCGCATAGCTGAGGCAAGGCTAGAACATTTAAACAAAACTTTGAAGTGGCAGGGATGCCAACAGAGAAATCAGCACAAAGCAGAGTCCAAGCAGCACCTGCAGATGGCTGCACTCCAGGACCCATAGGTCTTGGACCATCCTAGCTCCTGACCAGCATCCAAATGGCAGGCAACATCTTTGACACCCACATGCCCAGTGGCACAAGCCGGATCATCTTCCCTCTTCAAAGTCTAGAACCAAGAAAGAAATCCAAGGCAAAAGGAAATTCCCACTCCCAACTAAGACCCTTCTTCCCAGGCCCAGCTTGCTTTTGGCCAGATTATTGGCCCTGGAATGTCCACCTTCTCCCCTTAGGAGCCCCAAAGGTCAAGCTGGTCATACCAGCAAAGCACTGACACAGGATTTCTGCCCCATCAGTGCATGGGTGCCATTTCATGACAGAGCTTACCTGGGACCACATAGGAGCCTCACACACCTCCTCTCACCTCTGATGATGCTCAGGCTCTAAATCTTGTTTCTTCCATAGGAAAAGTTAAGGATTCCACAAGACTGGGAAATTTTATTCTTTAACATTCCCTGCTACAGAAATCAATAAAAGCAGCTCTCACATACTCCACTTTATAGAAATATAATAGGCTCTAGAGAATATCACATAATCAATCACCCACTTCATCGTCTCTGAGAGCACCAAGGGTTGCAAAGGTATGATCCCTGACCGTGGGCTAATCCAAGAGCAGCTAGCAAGCTACTCCATTCCCTAAAGTCAGAATATCCAGGGCAAACTTCACACCCAGCCACTTAACGATGCCGTTGCCTGCTCTGTTTAATTAGAAGTCATGTGACCATGAATGGGCCTGTATTTTTTCACCATTTTCAGTGATGAAATGGAAGGGATTAGTGGCTCAGCAATCCCCCTCCTGCCCCAGGAAAGATGTGAGCCAGCAGTTCTGCCATGAGCAAAGGCCACTTTGGTCTGATTCTTTGCTCTAACCCAACAGGATAGCAACCCATCCCCACCATGAAGCAGAAGGCAAGGATCAGCCTCATCACAGCACTTCCCCCTCTGCTTGCTGTGCAGCTCCCGTGGGCTCCCTGGAAGGGATCAGACAGGTTCTTCTCTTCCTCCCTCATAGCCACTCATGCAGAGGCTTGCACATCCCATACATTCAGTATGTACCCTTGGTACAGAATTGAATTTAGTCAAATAAAGGGTGACTTTAAATTTTCATTTGAGCTATGGTTAACTCAGAGCTGCCCATTCTGTCTTCCATTCATTTACAAATTTGCAGAGCATCTGTTGCAGCCAGGCACTATGCAGACACCAGGGTTAGAGCAGTGAGCAAGACAGACAAAGTTCCTACTCTTACAGTTTACAGTCCAGTGACAGAGGCAAACAGTTGGCCAAAAGTTGCACATATATGTTAATTCCTGGCTGGATAAATTGTCAAGGGAGAAAAAATGGGGTGAAGAGCAAAGGATGCTCAAGACTGAGCCTCGAAGAACTCTGATGTCTAAAAGTTGGGTAAAGGAGACTATAAAAGAGCATCACAAATGGCAAGAAAAGAAGCAGAAATGTAAGGCATGGTGGAGACCAAGAAAGCAACATTTCAAGGTAGAAGGGTACTAGGCAATATCAGACAAGCGTCCTTGGATCTAATATTATGGGAAGTCATTCATGATCTTAGTGAGAACAATCTCTGTCAGGATAGAGGCAAAAGCTCTGTTGATGTGGGCTGAGGAGTGAATGAGGGACACTGATTGTACACAACTCTTTCAAGAAATTTTGCTTTGAAGGTGGGAAGAGATCTAAGATGGTAGCTGGAGGGAGATGTAAGTTTCTGAAATATTCTGCTTTAAGTTAGGAGAAACTAGGTCATTAGAGATTTGTGCAATAAACCAAGAGACAAAGAGTAATGCACATGGACATTAATGTTGCCTAGGATAATAGCCAAGAGTGGGCAAATATGGCCAAGAGCCTCACCCATCATTACTGAGGCAGAGGGACCAAGAGTTTGGCAAGCGGCAGTAATGAAGAAGTGACAAAACTAGGTGGCAAAGCTTCAAAGCAAGAAGGGGTTTTACAAGACACAGGAAGAGCAGTGGTGTGGAAGGGTCAGTGGTGAACAAGGAGAACGCCAGGCCTAACCCCTGAGGTACATGGGGTAGGGGAGAATGGCAGCCTTTGAATAGGGGGAGATATGTGCAGATCAAGGCAGGTGAGCTCCATAGAGGTCAGGAGAAGTTTTGGGAGAAGGTCAAAGCGTGGGGAATGGGGTTGGGATCAAGGACTCACAGAGCAGTAAGATGTTCACAGCACTAAAAGAAGAGAAGATCACAGTAGGAGCTTTTTAAAGACACCATAAAAATAACCAGATATAAATGATCTCTTGGTTATCCAGGCCACTCTGCCTCCTTCCAAGATGCAAACAACCAAGACTTGACCCTACGCCCTATACCTGCATTTGCTTAGGCATTTGCACAGCACCAGGAACAGAGGGAACATTTCCAAGAATAAGTTTCCCCTTTGCACTTGACTTTACTCTCAGCAAAGTAGCTCAAATTAATATTTCAAGCAGCTCTTGAACTCTTGCAATGTGCAGGGCACTGCAGGGAAACAAAGTTAAGGAAGGTATAGCCCTTGTCCTCAAACAGCTCATTGTCTAGTAGAGAAGACGCATATACAAAATGCTAGAACTCGAGGCTGACTGAAAAGTGGGATCCCCAAAGGGACAGGGTAGTGTGTTGTCACGTGACTAAGGGACACTCCATGCAGTAAGTGGAGCTTAAAAGAGATGGATTACTCAAGACTGTTGGAATTTAGACAGCCAAGAGAGGAGATAGGAGGGCATTGTGAATAGAGGCAAGGGCACAGACCAGGACAAAAGAAGGGCCTGGTGGCATCAAAGGGCCAGGAGAGGTATTAAGGACTTGAACTGGGGCAATGAGGTAGAGTCAACTAAAAGTGAAATTTTGGAAGAGGTAAGGATAGTATTTCGCTTCTGTTAAGATGTGGAGTGTGATGGAGACAAAACTCATCTACAGATAACACAGATGCCTGGTTTGTGCTGGCCCAAAGGCCCTGATGCCACTATCTCTTGTTTTCTCAGAACAAGTTCCAGGGTTTTGTCTTTGACATCGTGACCAGACAAGCTTTTGACATCACCATCATGGTCCTCATCTGCCTCAACATGATCACCATGATGGTGGAGACTGATGACCAAAGTGAAGAAAAGACGAAAATTCTGGGCAAAATCAACCAGTTCTTTGTGGCCGTCTTCACAGGCGAATGTGTCATGAAGATGTTCGCTTTGAGGCAGTACTACTTCACAAATGGCTGGAATGTGTTTGACTTCATTGTGGTGGTTCTCTCCATTGCGAGTAAGTGGGCAGCCACGTGGTGGGGAAGCCCCACCTCTGTTTGGGGGTCTTTGGGATGCTCTTCTAAAACCAAGGAAATAACCAACCAACTTTTTGAAATACGTGTTTATGTTAGAAGAACCCTGTATTTGCAGCTTTCCAAGAGTAGCAAGTTTTAAATGCCTTGGGCTGCTCTCTCAGTGAAATGAAGAGGTTCTCTTGGCCTTCTCCATGCTCACACGAAGTGTTTTCAGGCACCTACTGTGTGCACAGCCCATGGCTAGAACCTCAGGGGCTGACAGGGAATAATCACTCCTCCTGAGAACCTCAGACCGTAAGATGATGGAGTCTCCAGCAGACCTTACTTTTATGGTCTTGAAAGCAGGGGGCAGGTGGTGTAATGTGAGATGAGTCTTAAAATCTGTTCTGGAAAAGGAGATGGATTGCCATCAAAGTGAGGAAAGACTGGGAGGAGATTGTTCAAAGGAGATTCTCAAGTCTAGGGTCTGGAATTAAATCTCTACACAACTGTTCTATTAGGGAACATGAGTGATAGAAAAGTCAAGATATCAATGCCAAGAAATTAATACTGTTAAGGCCCAGTCTGGGTGTTGTTGAATGACAGGCCCAGTATTTTGGGTCCAGTGGTGTTCAGAGAAAATTACTAGAGTTAGAGGCTAACATGGGGAAATGGAGCTGCCTTGACTGAATACTTCCTAAGTTCTGAGCACTGAGCTGGGCTCCATGAATGTTCACAGCAGCCCTGTCAAGCAGGTGTTATTATCCTTATTTTATACATTTAAAATTGAGACTCAGAGATCAAGAATATTATGCAGGGTCACAGAGTTTCCAAGGGTTGAAGCTGAGTTTAGAACAGGCTCCAATCTTCTAATGAACTTGCTGCCTGTCACCAACAGCCAAGAGACTGTCCCTGGGCTTATGGGTTAGCTGAAGTTTGACCCTATAAAGGCCAAATCATGCTCACCAGCTTCACCTGGCATTATAAATGTATCTTGATTGGTTATATCCTGACCTGCTTACTTTTCATGTCGGGGTGTAGCTGTAGATTTAATTTGCCACTGATTTAGTAACAAGAGCTGAGAGTATCTAAATACAACTTAACAGCATTTCATCAACCTCCCCACCTTGGTGCTGAAACACAGCCTTGGGTTGCGTGTATTTCCGGGGATTTCAGTTTGTTTTTGTTCTTGTTTTATTTTGATATTGCTGCTCTGTTTTTTAACACTTTATTGAGGTATGATCCACATGTAAAAAGCTATACATGTTTAATGTACACAACTTTATGAGTTTGGAGATAAGTATACACCCATGAAACCATCACCACCATCAATGCCAGAAACATGTCCATAACCTCCAAATATTTCCTCCTGCCTATTTATTTATTTATTGGGTGCTAGGAACACTTAAGATTTATTCTCTTGGCAAATTTTTAGGTAGACAACACGGTATTGCTAACTATAGGGACTATGCTATAAGTAGAGCTCTAGGATTTATTCATGTCACATAACTGGAAGTTTGTACACTTTGACAAGTACCTCCCCAATTTCCCCTCCTCCCAGCTCCTGGAAACCACCATTCTATTCTCTGCTCCCATGAGTTTGACTATTTTAGATTCCTTATATAAATGCAATCATGTCGTTTTTGTCCTTCTGTGTCTGGCTTATCTCACTTAGCATAATAGGAGTTTTTGTTGGTTGGTTGGTTAGTTGTTTTCATTTTTGTTTTGTTTTGTTTGTCTTCCAAAATGAAAGATTCCAAAGGCTATTTTGATTTTATATTAAAGAAGGGAGCTTGTTTGTGTATCAGTAATATAGAAATAAAACATAGCCAAGTACTTAGTTCTATGATGGGGATATCTTAACTACCAGTTCCTGTGGCTTACAGGGTAGCACCATCCGCTGGGGCAGGAATCCCTTATAGAGCTGGAAAATAAGCAGAAAAAACCCACATATCAATCTTTCTACCCTCTCTCCTTACCTCCCTTTCTTCTTTCCCACCAACAAGAGGTGATTGAGTATCTAATTGTTACAAGTACGAGGAACCATTCTCCCTTAAAATAAGATCCTGGAGCAAATTAAAATGTGCTGTAGACCAGTTGTCTGGCAGAAAATTTTACCAGAGTATGTAGACAAATAATGTACCCTACACGATGGGAACATGTCCTAGTTAAACTGGAAATTCACAGAAGGTCTTCCACAAAGATTTTATAATCCAAAATTATAATTATTTCCCAAGTCCCCTTCTACTTTCTTCCTCTTTCCAACACAATATTGTGACCCATGTGCATAACAACAAAGAGCAAGTAGGGGAGAGGGCCCAGGGAACCTGGCAAGGAGCGAGAGCTTGCTGCTACATCCCTCACCATTTTTCCTGTGAGATGTTGAACCCTCTCTTACCTAAAGGCCTTGCCTCATCTTTGGTGGAGGCCTTTGGTACAAATAAGGCTCCCCAGGTCCCCAAGACCAAAAATCAAACATCTTCAAGTTAGACATCATATAGAGGGACATATACATTCTTTATATTTCCAACTTTGTTCATAGTTGGAGTTGGGGGCAGTAACTGACTCCCCCAAAAAAACCTATCTCCAGGCCCTAACATAAAGTTCAAAAACAGCTGGAACCGAAAAAAAAAAAAAAAAAACAAATTAAGGCATTGTGCAGACAGCTGGTCATTCAAATCCCTTATGCTTTAGCTGTCAGCCAGTTGCCCGAATCCTCAGGCCCCCTTGTAATTAACACCAGGCTCCGTACAATTAGCCACAGGACCGGGTGCCAGAAACAGAGTCAATCTTCACAGTAGGAGGTAGGGAGAGGAATCATAAGGGGTCCTGGCCTTCACAGAGCACAGGACCAGGGTGGAGGAAAGGGCATGACCCAAAGGCTGCAGTATCAGGGAGGAGCAGGGTCTGTGTCATAGGAAAAAACAGGCCAAAGACTGTGAGCCTGTGGAATAAACTGCAGTCACTTCCAGCTGAACTTGGCTGCAGAAGATAAGTTTTTGACTGTGGAGGTTGACCAAGAGGATATTCTAGGCGGAGGCAAAGATAACATGGTAGGAAGGGTAAGACGGAGGAGGAAGAGAGGACTTATGCAATGGGCATTTACTGAGCCCCTACTTTGTGCTACCTACCATGCCAGGCTCTGAGGGCCCAGAGTCCTCAAATGTGGATGCCCAAGTGGCAGTCTATACCAAGCAAAGCAGAGTGTCATCACAGCCAACTTTGTCACGTCTCATGGCTGGAGATACTAATTAGATAATTTCTTCCTTCTTTTAAAGGCCTGATTTTTTCTGCAATTCTTAAGTCACTTCAAAGTTACTTCTCCCCAACGCTCTTCAGAGTCATCCGCCTGGCCCGAATTGGCCGCATCCTCAGACTGATCCGAGCGGCCAAGGGGATCCGCACACTGCTCTTTGCCCTCATGATGTCCCTGCCTGCCCTCTTCAACATCGGGCTGTTGCTATTCCTTGTCATGTTCATCTACTCTATCTTCGGTATGTCCAGCTTTCCCCATGTGAGGTGGGAGGCTGGCATCGACGACATGTTCAACTTCCAGACCTTCGCCAACAGCATGCTGTGCCTCTTCCAGATTACCACGTCGGCCGGCTGGGATGGCCTCCTCAGCCCCATCCTCAACACAGGGCCCCCCTACTGTGACCCCAATCTGCCCAACAGCAATGGCACCAGAGGGGACTGTGGGAGCCCAGCCGTAGGCATCATCTTCTTCACCACCTACATCATCATCTCCTTCCTCATCATGGTCAACATGTACATTGCAGTGATTCTGGAGAACTTCAATGTGGCCACGGAGGAGAGCACTGAGCCCCTGAGTGAGGACGACTTTGACATGTTCTATGAGACCTGGGAGAAGTTTGACCCAGAGGCCACTCAGTTTATTACCTTTTCTGCTCTCTCGGACTTTGCAGACACTCTCTCTGGTCCCCTGAGAATCCCAAAACCCAATCGAAATATACTGATCCAGATGGACCTGCCTTTGGTCCCTGGAGATAAGATCCACTGCTTGGACATCCTTTTTGCTTTCACCAAGAATGTCCTAGGAGAATCCGGGGAGTTGGATTCTCTGAAGGCAAATATGGAGGAGAAGTTTATGGCAACTAATCTTTCAAAATCATCCTATGAACCAATAGCAACCACTCTCCGATGGAAGCAAGAAGACATTTCAGCCACTGTCATTCAAAAGGCCTATCGGAGCTATGTGCTGCACCGCTCCATGGCACTCTCTAACACCCCATGTGTGCCCAGAGCTGAGGAGGAGGCTGCATCACTCCCAGATGAAGGTTTTGTTGCATTCACAGCAAATGAAAATTGTGTACTCCCAGACAAATCTGAAACTGCTTCTGCCACATCATTCCCACCGTCCTATGAGAGTGTCACTAGAGGCCTTAGTGATAGAGTCAACATGAGGACATCTAGCTCAATACAAAATGAAGATGAAGCCACCAGTATGGAGCTGATTGCCCCTGGGCCCTAGTGAGAACACTCCAGCCTGGATATGTTCAGTTATGATGCGTCCTTCTGCTCTGTGTTAACTCTTTCCCATTGCAGATCACACCCAGCTACAGCCTCACCAATGCATGCCACTGGTCACAATGTCAGAACTGGGCAGGGAATGCAACTGGTAACCACCTTTGAAAAAACCATCATACACAAATAGGAGTCAGAAGCTAAGAGCACTTCCACTGTGATTTCCCTTCTGAATTTCAATATCAGATCATGGGTTCTCTTACTCTCCAGAAAATATCCCTTGTCCTTTTATTTTTGTTGTAATCAGATTATATTTACTGAGACAAACTTCTCAGGACCAGAACTTCCAAAGATATAGAACAAGAACATTACTGAAGGTCCAAGGTAGTCCTCTGTGGAGCACCATACAGAGATTGGCAATATTATTTAGGATTTTGCATGACTGCATGTGAGAGCTGTCGGACAACTGCTCTGACCCAGGGTAACACCTGTGGCCTATGTCATGAAAAGCCTTTGAGATATCCATTAAAATTAATATTTTTAAATGTATGTCCACACCAGCGTTATATCATAAAGCATCATTTTATTTTTCTCTTATTTCTGTGATGCTGACCTGTATCATGAGTCACTTACCCCTCACTTTCTGGGATCCTGATATTTTTCAGTTTGAGATTGAGGCTGGGTGGAGCAGGAAAGAGCACTGTCCTGGGATCTGGAAATGCCAGTTGGAATCCTAACTTTTCCACCAATTGTCTGGCTGACCAGGGCTAAAGTCTTTCCCCTCTTAACTTCAATTTCCTCACTTGTAAAATTGGTGGGAAGGGAAGAAAATGAATGCTTTCTATAGACTTCGCATCTCGTGCTGTAGAACTCCATGGTGCCTGCCCCATTTATATGGGAAAAGCACTCTTTTCTGAGGCTGAAATGGCTGGACCTTTCTGTTGTGTAGCTAAAGAGGCAGCTTTTGAATACATTCCAGAGTCTGTTCACATCTCATAATCAGACATTAAGGAGGAGAGTTTTTACATTACCTGGCGTCCAGAACTCCATAGTTAATATAATCAGAAGAAAAAGTATAATCATGAGAATGATGATCCAGTAGATAAAAGGTATGGCCAGTAGTTTCTTTCATTCATCCTGCAAATGTTATTAATTACGTACTATATGATGGGCACTGCTCCAAGAACAAGAGATAAAATAGGAATTAAGCCCCCAAAAGATTAATTGTATGAACTGAATGGAGTAAGAATTGTCAGAGGAGTTTATTTAGGCCAAAGGCATGTTGCAGATTTTGAGATGAAGAAAAGCAAAGACTCCCTATAACAACATAGGACATAGGGATTGTAATGACAGTCATGATCTTCCATTAACTCTTGGCCCATTCCCAGCCACAATCACAAAGATTCAAATATATAGCCAATTCATTGTCTATATATTTGATCATTTCATAAGATTTTCCATTTGTATAATTACAAAGCACAGTGGCTGATACATCTCACATGCCTCAAGAATACAGATTTGCCGAGGCCTTGCTCTCCCTTTTCTGTCACTATTAGCCCATAAGAAGTCAAAGAGGCTCTCAAGATGCTTACTGGGAGTTGGCAGCATCCACAGATGCAGGTTAATAAGATGATGATGGGATAGCAGAGGTACCAAGCCATCTCAAATCAGCTATTCTCTGAATGCCTGATTTATTCTAAAAGATGAGTTCAACCAAATAACTCTTCCTCTCTCGTACCACTGAA CCCGGGAGC137 Human Nav1.9MDDRCYPVIFPDERNFRPFTSDSLAAIEKRIAIQKEKKKSKDQTGEVPQPRPQLDLKASR amino acidKLPKLYGDIPRELIGKPLEDLDPFYRNHKTFMVLNRKRTIYRFSAKHALFIFGPFNSIRS sequenceLAIRVSVHSLFSMFIIGTVIINCVFMATGPAKNSNSNNTDIAECVFTGIYIFEALIKILARGFILDEFSFLRDPWNWLDSIVIGIAIVSYIPGITIKLLPLRTFRVFRALKAISVVSRLKVIVGALLRSVKKLVNVIILTFFCLSIFALVGQQLFMGSLNLKCISRDCKNISNPEAYDHCFEKKENSPEFKMCGIWMGNSACSIQYECKHTKINPDYNYTNFDNFGWSFLAMFRLMTQDSWEKLYQQTLRTTGLYSVFFFIVVIFLGSFYLINLTLAVVTMAYEEQNKNVAAEIEAKEKMFQEAQQLLKEEKEALVAMGIDRSSLTSLETSYFTPKKRKLFGNKKRKSFFLRESGKDQPPGSDSDEDCQKKPQLLEQTKRLSQNLSLDHFDEHGDPLQRQRALSAVSILTITMKEQEKSQEPCLPCGENLASKYLVWNCCPQWLCVKKVLRTVMTDPFTELAITICIIINTVFLAMEHHKMEASFEKMLNIGNLVFTSIFIAEMCLKIIALDPYHYFRRGWNIFDSIVALLSFADVMNCVLQKRSWPFLRSFRVLRVFKLAKSWPTLNTLIKIIGNSVGALGSLTVVLVIVIFIFSVVGMQLFGRSFNSQKSPKLCNPTGPTVSCLRHWHMGDFWHSFLVVFRILCGEWIENMWECMQEANASSSLCVIVFILITVIGKLVVLNLFIALLLNSFSNEERNGNLEGEARKTKVQLALDRFRRAFCFVRHTLEHFCHKWCRKQNLPQQKEVAGGCAAQSKDIIPLVMEMKRGSETQEELGILTSVPKTLGVRHDWTWLAPLAEEEDDVEFSGEDNAQRITQPEPEQQAYELHQENKKPTSQRVQSVEIDMFSEDEPHLTIQDPRKKSDVTSILSECSTIDLQDGFGWLPEMVPKKQPERCLPKGFGCCFPCCSVDKRKPPWVIWWNLRKTCYQIVKHSWFESFIIFVILLSSGALIFEDVHLENQPKIQELLNCTDIIFTHIFILEMVLKWVAFGFGKYFTSAWCCLDFIIVIVSVTTLINLMELKSFRTLRALRPLRALSQFEGMKVVVNALIGAIPAILNVLLVCLIFWLVFCILGVYFFSGKFGKCINGTDSVINYTIITNKSQCESGNFSWINQKVNFDNVGNAYLALLQVATFKGWMDIIYAAVDSTEKEQQPEFESNSLGYIYFVVFIIFGSFFTLNLFIGVIIDNFNQQQKKLGGQDIFMTEEQKKYYNAMKKLGSKKPQKPIPRPLNKCQGLVFDIVTSQIFDIIIISLIILNMISMMAESYNQPKAMKSILDHLNWVFVVIFTLECLIKIFALRQYYFTNGWNLFDCVVVLLSIVSTMISTLENQEHIPFPPTLFRIVRLARIGRILRLVRAARGIRTLLFALMMSLPSLFNIGLLLFLIMFIYAILGMNWFSKVNPESGIDDIFNFKTFASSMLCLFQISTSAGWDSLLSPMLRSKESCNSSSENCHLPGIATSYFVSYIIISFLIVVNMYIAVILENFNTATEESEDPLGEDDFDIFYEVWEKFDPEATQFIKYSALSDFADALPEPLRVAKPNKYQFLVMDLPMVSEDRLHCMDILFAFTARVLGGSDGLDSMKAMMEEKEMEANPLKKLYEPIVTTTKRKEEERGAAIIQKAFRKYMMKVTKGDQGDQNDLENGPHSPLQTLCNGDLSSFGVAKGKVHCD 138 Human Nav1.9AAGGTTCTCCAAGGCCCCACCAGAGCAGCTAGATACAGAGTGTCGATTGGTGCACTCACA nucleotideAACCCTGAGCTAGACACAGGGTGCTGATTGGTGTATTTACAATCCCTGAGCTAGATATAA sequenceAGACTCTCCACGTCCCCACCAGACTCAGGAGCTCAGCTGGCTTCACCCAGTGGATCCCGCACCGGGGCCGCAGGTGGAGCTGCCTGCCAGTCCTGCGCCATGCGCTCGCACTCCTCAGCCCTTGGGCGGTCGATGGGACTGGGCGCCGTGGAGCAGAGGGCGGTGCTTGTCGGGGAGGCTCCGGCCGCACAGGAGCCCATGGAGGGAGTGGGAGGCTCAGGCATGGCGGGCTGCAGGTCCCAAGCCCTGCCCCGCGGGAAGTCAGCTAAGGCCCGGTGAGAAATCGAGTGCAGCGCCAGTGGGCCGACTCTGCTGGGGGACCCAGTACACCCTCCGCAGCCACTGGCCCGGGTGCCAAGCCCCTCACTGCCCGGGCCGGCAGGGCCAGCCGGCTGCTCCGAGTGCGGCGCCCGCCAAGCCCACGCCCACCCGGAACTCCAGCTGGCCCGCAAGCGCCGGGTGCAGCCCCGGTTCCCGCTCGCGCCTCTCCCTCCACACCTCCCTGCAAGCTGAGGGAGCCGGCTCTGGCCTTGGCCAGCCCAGAAAGGGGCTTCCACAGTGCAGCGGTGGGCTGAAGGGCTCCTCAAGTGCCGCCAAATTGGGAGCCCAGGCAGAGGAGGCGCCTAGAGCGAGCGAGGGCTGTGAGGACTGCCAGCACGCTGTCACCTCTCACTTTCTTAGGGTCGAAGTCCTAGAAGTGAGAGAGCCGGCATGGGGGTCGCAGTTCATGCCTATGTTTATGGCTCTTTTGTTCCCATTTCATCACATTTTAAGAACTTTACTGACTGGATGTGAGAAAAGACCCATGCACTTTCAGTTTTGCTTTTGCAATCCATAACTATGGCCCAGTCAATTCCCTGTTCCTTTTTCTCTTGTTTGGGCCATGTGGCCACTTGCCTGCTACTTGCAGATCCCACTTCAGCAGTTCAGCCGGCTCAGCCTTATTGTCTTGCTTAATGTCTGGGTCTCAGTTTTAGAGACTGGGCTTCTTCTGCTCACTTGTTCCTGAACTCAACTTCCCGCCTTTTGCCAGGTCCTCCAGGAATGATGCCCTGCTTTGGTTTTGTCTATGCCAAAAGTTCCTGCTATACCCACAGTGGTGGTCATCTCTTCTGATCTTCACAGCCAATCAGCTCCCAAGGCCCCTGACCTCAGCTCAGCTTTTGTAGATCCTTATGACACCATCCTTTAAGACTGGAATCCTAGGGCAGGCTGTTTTATTCCCGCCTCCTGAGGCCTTTCTGAGGATCTGTGGCTTGTCTCTGTCCTGAGGGTGAAGATGGATGACAGATGCTACCCAGTAATCTTTCCAGATGAGCGGAATTTCCGCCCCTTCACTTCCGACTCTCTGGCTGCAATTGAGAAGCGGATTGCCATCCAAAAGGAGAAAAAGAAGTCTAAAGACCAGACAGGAGAAGTACCCCAGCCTCGGCCTCAGCTTGACCTAAAGGCCTCCAGGAAGTTGCCCAAGCTCTATGGCGACATTCCTCGTGAGCTCATAGGAAAGCCTCTGGAAGACTTGGACCCATTCTACCGAAATCATAAGGTACTTCATTGGGCGGGGGGGGGGGGTGGGGGTGGGGGTGTTCTAACCATGCAGTTGTAACTGGTGTTACAGGTTCTCCAAACACCACTCTTAGGGTTTATAGCAGGCAGGCTGTGCCTCTTCTTTGCTGTCATCCCCTGGCATTGCCTTGATAGTCCTGAAAACCCCTTTCAGTCCTCATTTTTTCTCTGCAATGCATGTATTAGAGGAGAAGATGAGGATGAGGGACTGCAAAAGGATAAAAGGAAAACAGAGAAAACACAAAATGAAGTCTGGAAAGAGGACTGGACTAGTAGTCCCAGAAGTTAGATACAGGCCATTTCAATTGATTGGCTTTATGACTCTGGGCAAGTCACTTTACTAATCTTCACTTTTAAAATCTGTATAATAGAAATAATGCCTGATGTTCCTACCTTAGAGCATAGACATGCTGGGAAAGCACTTTATCACTATAATGCATTATACTGATACAAAGGATTAATATCATGGTCCAGAAATGCTCATTATAGCAACAAGATTTTACCTTAAAAACTCTTGGATAATAGGCTTTCTAGGCTATTAAATGAGACTAATAGGGCTCAAACCAACAACTGATGCATAGTAGGAGCTCAATAAATGAAACCTAAGAAAATGGTAACCAAGTGGGCACATGCAGATTCTATGTAAACTCAATGCAAATATGTGAAAGCCTTGCTATTTTATCTGCCTGGATGCAGTTCTTGCAGGGAAATTCTAGTAAAGGTTTCTTGAAGGGGCTCCCGAGTGAGGGAATGTAAACTTTGAGCTCCCACATGACTGCTCTAGAGCCTGAGGCTGTGAGCACACTTGGCCCTTTCAGGAAAGGCTCAGAAGATTTTGCCATCATCACCTCGCAGTGTGATCCAGCAGAGGTTGGAAACCCCACCTTGCCATACTGAGGTTAGAGACCTAGGAGGCTGGGAGGTTTGGGGAAGCCCTAGAAAATATGGGATGCTGAGAGCATCTTTGGCTGGCATCCATAAGAAACTGACCTTTCAAAGAAAGATTGCCTGATTTGCATGTACTTCAATTGGGATAGACTTAGAGTTGCCAGGCCAAATGAGGACCACATATACAGAGGAAAGTAGAGTATAAGTAGGAAGGACAAGCAAGGGGGAAAGAATTGAAGGAGCAAAAACACTCCAATGTTTCTCCTTTTTCTGATGCCCCAGGATTCCAGGGCCTTCTTGGAGGTATAGTTGTTGGAGGTGAGTTAACTGTAGAGGTCTCCTTCTGCATCAATGCTGGAGAGGAGACGCATGAGTCAGGCAGCAGTTTGGTGTGCAGCTGACAGTCCGGGAGAACAGGACTATTCCAGCCTGGGTGGTAATTCTAGGACCACCACTTCTCAGCCTGGGTCCTCACTTTAAATAAGAAGACACGATATCTGTTTAGAGCTCTGCACACCCATGATCTCACATGACCAGTGCCCTCAACAGTCATGTGAGGTATGTGTGATCATGATTAGCACTCTACCTTACAGATGAGGAAACCAGAGCCTAGCTGGGAAAGTGGCTTAAGCCAGGTCAAGCAGCCAGTGAGTGGTGGAGCTAGGATGCAAACCCAAGGGTGCTTTTTATCATCACAGGCTACTTCTCTTTGGAAGCCCCATTTTGAAGAGCAACAGGGGAAAGAAGTAGAGAAGTGTCAGCAAGCAGGCAGGAGGAGCTAGTACCTATCATTTTAATCATTAAAACCCTGGCATGTAGAGTTAAATACACCATTAGTTAAAATATTTAAAGCAGTAAGAAGAGGCATCAATAAAATATCAAAATCAAAGGCACAGAAGGAGAGCCATATTGTACCACTTATTAATGTGGGGTGACTCATCTCAAAGAGAGGTTTGTTTCTGCCCCAAAATTGTATGTCTGTTCAAAGGAAAGGCGCAGAATTTCCAAAGCAGGTCTTGGGCTCCTGGCTCATATTTTTAAAAAACCCCGTGGCTCATTTCATTATTATGGGGGCAAAGAAAAATACCCTAGCACAGTTTTCCCTGCAGCCAGATAACCATCAGACCAGTCAATCCCAGCAGCTCATTAAAGGAAGGTTGATGGTTGGCAGTGGCTGACGTTCTGGCACCTGAGCCAAGGTAGCACTCACAGTGCTAACGTTGGGCCTGGGTGCCGAGGTCTGTCTCTCCTGCCTGAGAACATGCTGTATTACATGGCCAAGTAGTACTTTTTGCAAATCTGAAAGGAGCCATCAGTTGGGGAGAAAGGAATGACTCTTACTTTCTGAAGGCTTTTGTAACTGATAGCCATGGGGACAGATATGGGGACAGCTAGATTACAGTTACATGGAAAAAGGGAGGTCTGGAGGTGGATGTTAGAAGCATGTTAGTGACAGGTTGTTGTCCAGGGGCTGCTTCATTCCCAGTGCCCAGGGAGTCATGCTAGGAGAAAATGGGCAGAGGTTCAGTGTTCTTTAATGGGACTCCACTGGGCACTTTGTGGGGGGGCGGGCATAATGTACCATTGGCAAGTTTGTCCTGTTTGGCATCCCTGGGTCTTCCCAGTAAAAGGCCGGTAGAGCCCATAGTCATTACAGCAGACAACCAAAAATACTCACACACATTTCCAAATGCCTTCTGTAGGGGCAATGCCCACCTCCCCTGCTGGTTAAGAACCACAGGGAAATTTGCCCCTATGTCCACAGAGGTAAAGAAAGAGCAAATCCAGGAAACTTGGTGACTTTTTTCTTGAAGATCATCAAGGTTAGAATTGACTGGTTATTTCACACAGCATTTCTCAGACCTGTGGCATAGCAAACATGGACCCTTTAGGTCACTTATTCTAAGATTCATTGCAAAGTTTTCGTTAATAATCAAAATAATTTAAAATAATCTGCTGAATCTAAATGTCCAAGCTCAATTTCTGCCTCTTCAAGTTCTCTTCTAGTCCCTTTCCCCGTTGATACGTACCATGGAGTACAATATTAAGGGTTTTCTTTTCCCCAGTTATTCCTCTATACTCCTAATTTTGGTATTTTCAGGTCCTGTGTTTTCCATCTCTGATTCTTCTGGAGTATCCCATTGGGTAAATATTCCACAGCCCAACAGTTTATGTTATAATTTTCTCTAAATGAATTCATGATAAATATTGTTTTGTCACTGCATTTAATTCCTGTTGTGTATGTGTGTGTTTTCTTGTATTGTTTTTTTTGTTTTGCAGACATTTATGGTGTTAAACAGAAAGAGGACAATCTACCGCTTCAGTGCCAAGCATGCCTTGTTCATTTTTGGGCCTTTCAATTCAATCAGAAGTTTAGCCATTAGAGTCTCAGTCCATTCATATCCTTTCATTGCACCTTTTACTAAGTCCAGATCATTTGAAAAGTGCACGTGCCCCACGGTGTTCATGTTATTGGATGGAAGCAAATAGCTAATTCTTTCTACAAATACTGGCTGAGTGCCTACTGCTGCAGGCAGGGACTGTGCTGGATGTGAGCAAGATGTCGTCTCCTTCTCTGGGGTCCTATGGCCTCAGAAGGGCATAGACTAAAAGGCAGGCAACTTCTTTAGTGAGATTGGTCGTGTGAAAGTGGTAGCAGGGAGAGGCACTGCTGTGCTTTAGAGGCATCAAGGATGGCTTCTTAGAGGAAGGTGCATCTAAGCTGACACCTAAAGAGTTGTAAACTGGCAACCTGTGCAGTATATTGAGTTGATAATGTGTTGTGTTGGACCTACACAGTATTTAAAACTTTGAAAAACACTTTCTAACATGAATACTCCAGATTTCCAGCTTTTCTTGAAAGATGGAAACTGGCAAGGGTAGGTTCCCATTTCTGTTAATACCTGACAAAAATGGGTAGAGTAAGGAATTGCTGCCTCTTTGGAGGGGGCATGTAAGCTCTGTCTCACCACCTCTGCACCCCTCTTGTGGCTGAACACCAGCCCATTTTACCCATTTATATGCCTTTTCAGGCTCTTAAAACTTAGGGTTTGCAATGTCAGTCTTAAAATGTAAAGGCAGGTAGGAATTATTAAGCATTTCTGCTGTATGCCAGGGTTGGGGGAGACACTAGGAAGTTTTAGACAGGGAAATGAGAAAAAGATATGGGTTGGTAGATTTAGTAAAATATTGAGGAGAATGGCAATGAATAACTATTTGGGTTTGGTTCCCTAGAAGCAGAGCCTGATATGGGCATTCTTATTTAAGTGATTTAACAGAGGAGTGCTTTCTGGAGAAGGAGAAAGGGAAGCAGAAGGGGGGAAAACAAAGAGCCAAGCAAGGATATGGTCTCAGCTGAGTCACCTTCATTCTGATCCCACGAAGAACTCTGGAGCATGAATTGCACCACAGAATTGTCCCACTTTGAAGCAAGGGCCTAAGTCAGTCAGACATGTAGAAGGGGGTGTGTAGCTCTCCAAGTGAGGAGGCTGCCATTCAGCAGGGGACAATTCTCCACAGGAGGGGACAGTTGTGAGTTGTTCTCATCCAACATTCACAGTCAGAAGATAGATGCACCTGCAGAAAAGGGGACCATCAGGGGCACCCCCAGTCTTACCCAGTAGCTAATGATGCATGCCATCCTGCAACATATCTGCCTTAATGGATGCTGGCCACATTGTTCAGCATGTTCATTATCGGCACCGTTATCATCAACTGCGTGTTCATGGCTACAGGGCCTGCTAAAAACAGCAACAGTAACAATACTGACATTGCAGAGTAAGTATTTTTCCTTCACTCACCTGTCCTAAGTTTTTAAACAAATATACCTAAGACAGTTAATGATTACACTGGAGAGAAAGAAAAAGTCACTAATAATTCTAACACATATGTTGGCAGAATCAGTTTTATTTTTGACCTTTATTTTTTTACAATTTGTAATGAACAGCACACATATGTTCACATTTTAAGTGGTTTTCTTTGTGTTTACATAAGTTTTATAGGTATCCATTTTGATGGTTATATAATATTTTATCAGGTGAATAAACCATAATGTACTTCACCACTCTAGCATTTTAGTAGATTGTAACATTGTCATAAAAATCTTTTTGCATAAAGCTGTATTTATGTATTTACTTATTCTTTGTTTTTGTTTGTAAAAAATTGTTATGGGAGTTCACTATGCCAAAAATAAAGCTATTCTTTTTTTATTTTTTTTTATTTTTTGAGACAGGGTTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCACTATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCGATTCTCTTCCCTCAGCCTCTCAAGTAGCTGGGATTACAGACGCCTGCCACCGTGCCCAGCCAATTTTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCTTGACCTCAGGTGATCTGCCCACCTTGGCCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACGCAGCCCTATTCTTAAATTATTATTTTCTTGAGATACACTCTTAGAAATGGATTTTCTAGGCTGGGCGTGGTGGCTCATGCCTGTAATGCCAGCACTTTGGGAGGCCAAGGCGGGTGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCTAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAAATTAGCCAGGCGTGGTGGCGGGTGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCATGAACCTGGGAGGCGGAGCTTGCAGTGAGCCCAGTGAGCCACTGCATTCCAGCCTGGGCAACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAATGGATTTTCTAGATAAAAAGGTACATTTTAATGCTTTTAATGATATATATAGACAAAATGTTTTCCAAAAAGTTTGTATCAATTTGCTCTGCCATCAGCAGTGTATTCTAATAACCAATTATATCACATTCTTGCTAGCATTAAATTTTTTCTTCTCTAATTTATGGAGTTAAAAATATTAGCATTTCTTTAATTTCCAGATGGGTTAGACTTAAAACAATTTTAATTGACACATAATAATTGTACACATTTCAGGGTACATGTGATATTTTGATACATGCATACACTGTGTAATGATCACTTAAGGGTAATTGGGATATCAATCCCAATTGGACATTTATTATTTCCTTGTATTGGAAACATTTCAAATCTTCTCTTCTAGCTATTTTGAAATATACAATAAATTATTATTAACTAAATAGTCATCCCACTGTGCTATTGAACACTAGAATTTATTCTTTCTGTCTGTTTTTTGTACTCATTAATCAACTTCTCTCCATTCCTCACCTCTCACCCAGTCTTTCCCCATCTCTGGTAACCACCATTCCATTCTCTACCTCCATGTGATCCACTTTTTCAGTTCCCATATATGAATGACAACATGAAATATTTGATTTAGACTCTTTTAATGTTTTCATTTCTGAATTGTGTGTTTCCATCTTTGCTTCTGTATCATTTTTATTTCTTTTCTAATCAGTTTGAGTTCTCTGTATAACCAAAATATTAACTTTTTATCATTTGCATTGGCTTTTCAGACATATGAATTAATGTTTTAACTTAAAAGTTCTATATTTGTATAAATATTTTCTTATCAAAAGTTCCAGTTTTTCTAGTTTAACATAATTACTGCTTTAATCTTTCAGGAACATATTGAATGCAAATTGAATAAATGAGACCATTTTTTCTAACTCCTATTTAATTATGCAATATAAACCACTGCAATCGATATATAGACCATTTCAATTACCCCCCAAAACGTCTCATGCCTCTTTGCAGTTAATCCCCGATTCCCACTCCCAACCCCAGACAAACATCGATCTCATTTCTGTCATTCTAGACTTGTTTTGCCTTTTTTAAGAATTTCGTATACATTTCATATGCTATGTATGATACTATATTTTTGTATCTGGTTTCTTTCATTTAGCATAATGCTTTGAGATGCATCCATGTTGTTGTGTATATTTCTACATTTTTTTTTTCTTTTTGTTGCTAAGTTGGATTCCATTGAATGGATATAGTTTTCCCATTTATCTATTGATGCACATTTGCATTATTTCCAGTTTAGGGCTATTATGAATAAAGTTGCTGCGAACACTTCCGTATAAAAATCTTTTGTGGATATTTTTGCATTTCTCTGGGATAAATACCTAGGTGTAATTGCTGGGCCCTGCGATAAGTATATATGTAACTTTCTAATAAACTGCTAAACCTTTTTCAAATTGACAGCATAATTTTACATTCATATCAGCAATATATGAAAGTTTCAAAGTCTTTATTAATTTTTTCATTCAATTTGCTTTGATTTTAATTTGCTCTTCTTTTTCTAGCTTCTTAAGTTGGAAGCTTAAATAGTTGGTTTTAGATTTTTCTTCTTATACAATTGTATAAAGCTATGCATTTCTGTCCAAGAACTGCTTTAGCTGCATTCCACAGATGTCAATACACTGTGTTTTTATTACCTTTTGGTTCAAAATATTCCCTACATTTTCTCATGATTTCTTCTTTGTCCCATATGTCATTTAGAAGTGTGTTTTTTAATATTTCCAAATATTTTATACCTTTCTAGTTGTTTTCTGGTTGTCTGATTATTATCTAATTTCAGTTCAGTTATGATCAGAGAATACACTCTGTAAAATTTCAATCTTTTGAAATAAAGAGAAACATCTTTTATAGTCTATCTTGGGAAATGTTTCACATGCACTTGGAAAGAATGTGGATTCTGCCACATTCTTAAATACTCCTTAAAAGGTCAGTTAAGTTAAGGTGGTTGATAGTGTCATTTAGATCTTTTGTACCTTTACTGATTTTTACTTAGCATTTTTATGAATTACTGAAAGAGAGGCTTGAAATCTCTATCTATGATTGTGGATTTGTCTGTTCTTTCTCTCTTTTTGTCAAATTTGGCTTTGTGTATTTTGAAGCACTATTATCAGGTACATACATATTTACGATGATTATTATTATCCTCCTGATGTATTCATTCTTTTATTATTATGGAATGTCTTCTTTGTCTCTAATAATACTCATTTAAATATTTGTTTTGTCTAATATTAATATAGCTACTTCAGCTTTCTTAAGCTCTTTGTTTGCATGGTATATCTTTTTCCATTCTTTTACTCTCAAACCTTCTGTGTCTTTGTATTTGTAGCGTATATCTTGTAAACAACATATAGTTGGTTTTTGCTTTTTATGCAGTCTATCAATCCAGTCTCTGACTTTTGATGACTAAAGTTCATTTACATTTAATATAGTTATATAATTGGATTTTGCTTTGCTATTTTTCTATTGGTTTTCTATTTATCTCTTTTTTTTTTTGTCATTGTTGTTGTTCCTTTGCTACTCCATTATTTCTCCATTCCTGCTTCCTTTTGTGTTAATCATGTATATTTAGTATTTCATTTTAATCCTCTGCTGGCCTTTTAGCTTTACCTATTTGCATTTTTTAGTGGTTTCTTCACATATTAAACAATGCATTGTTAAATTGGCAAAATAGAGTTAACGTTGAATTCCTTCAAGTGAAACAGGAACCATGCCACAGTTTAGTCCCCTGTTTGTTTTTTATCATATAGTTATGCTAATGTACACAATAAACCCAACAATACAGTTTATCATTTTTGCTTTTAATGGGCACATGCCTTTTAAAGACATTTTTTTTCCTGTAGATCCAAGTTATCTTTTGGTAGTATTTCTTTTTAGCCTGAACAACTTCCTTTAATATATCTTGTGGTTCAGTTCTGTTGGCAACCCATTCTCAGTTTATATTGATCTGAAAATGTCTTTATTTCATACTCATTTATGAAGTAGAGTTTCATTGTATATAGAATCAGTGCTTGACAATTTGTTTTTCTAGCACTTTAAGTATGTCATTTCCATGTTTTATGCCTCCTGTTGCTTAGCTCTCTCTTGCATGCTTCCAGGATTTCCTCCCTAAATTTCCAGCTGCTCTGCCAGCTCTGAACCCCACCCTCGGCCACCTTAAATCTTCAGGACTGCAGCCATTTGCCAGTGTTGGGGTGGGAGTGGGGAAGTTGGAAAACACTCTCAGACCAAAACAGTTAAACATTTTCATAATTCTTATGCATTGCAGGTACCTTCTTTTAAGGGGAAACTCTGCCCCAGTTTCTGCCTCCTTTTGTTTACCTTCCAGGGCCTTCAAATAATTGTCCTTATATATATATTTTCCAGTTTAATACTCATTATCTGTGGGAGGGTTTGCCCAGCTACTTCATGTCTTCATCACAATTGAAGCAGGCATCAATGGTCTCTTAATTTTGCTTATTATTTTTTTCTTTTCCTTTATTAAATTTAACAAACTTTCCTCTATGCCCGTGGATTTTGATTTATAGTTAGAAACACTTTCTCTAGCCTTGAGTTATAAAAGGACTCTCTCATATTTTCTTACAGTGCTTTTATAGTTTCCTTTTTTAAAAATTTAAATCCTTGAATAATTTGGAATTCATTCAAGGGTAGGGTGTCATAAATAGATCTGACTTCATAGTTCCGTCCCAGATGTCTATCCATTTGTCCCAACACCATTTGTTAAATAGTCCACCTCTTATCCTCTGATGTGAAATGTTACTTAGGTATTCAGATCTATTTCTGGACTTTCTTTTCTCTTCCATTGGTCTGCGTTTTCATATGCCAGAACCACACTTTTTAACTATCAAGATTGTCTAATGTTGTAATGTCTAGGAGTGCTAGAGGTTCCTTTTGAGAATTTTTATGGCTCTTGTTGATTTTTTCATGTAAATTTTATAGTCAACTTGGTTAGTTTTATTGACATGATGAAAGGATTTATAAATTAACCTAGAGGAAACTGACATCTTTATGAGGTTGGAACTCATTGAGAACATGACATATTTTTCATTTGTTAAAATCTTCTTTTGTGTTCCTCTAATTGCTTTAAAGTTTTCTTCATACAGATCTTCCACAATTCTTGTTAAATTTGTTTGTGAATTTTACTGTTTTTTGTTGCCATTATAAATGTGGTCTTTTCTTTCATTATGCCTTGTAACTAGTTGCCTTGTTATATAAAACTATATATATATATATATATGTATGTACATCAGTATTATACATATCAATATTTTATATATATATAAAATTGTGTGCTATTTTTGATAAAATTTGTTGTCAGCATTTGTTTACTCTTTTGATGACTTGTATGGCTCCTCCCCATCTTTCCCATGTATTAAATTTTTTTTTTTTTTGAGATGGGGTTTCACTCTTGTCGCTCAGGCTGGAGTGCAATGGCGCGATTTTGGCTCACCACAACCTCCTCCTCCCGGGTTCAAGCGATTCTCCTGACTCAGCCTCCAGAGTAGCTGGGATTACAGGCATGCGCCACCACACCTGGCTAATTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGTTGGTCAGGCTGATCTCTAACTCCCGACCTCAGATGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCAAGAGCCACTGCGCCCGGCCTTGAAATGTTTTTAATAGCATGGAAATGAGGTATTCTGGGAGAAAGAAAGAGAGAATTATCCAGTAAAGTGATTTATATCTGCAAATCTTTTGGAGAGGGATATAAGGAATTAGTGAAATGGGAAGGAATTCATTTATTAATAGCTTTTTCATTTTTCATTGGTAATCTTTTTCAGTTTTAAAATTTCTCTGAATATTTTGCTATATTCTTCTTCTCATTTATCATTTTGTTCATATGTTTTCTTTTTTCTGTGATTAGAATTGCCAGAGGCTTGCCTATTTTTATGTTTTTTTAAACTATATCTTAAATTCCATTTTAAAAACTATTCTATAGTTTTTCTATAACTATGTCTTAAATTCTATTTGTCTTAAATTCTATGTTTTTCTAAAACTATATCTTAAATTCTATTTATGTATTAAATTCTATTTTTATTTTATTAGTATTTTTTGAGACAGAATCTCGCTTTCTTGCCCAGGCTGGAGTGCAGTGGAGCAATCTCTACTCACTGCAGCCTTCGCCTCCTGGATTCAGGAGATTCTTGTGTCTCAGCCTCCCAAGTGACTGGGATTACAGACATGCACCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTTGCCATGTTGCCCAGGTCTTGAACTCCTGGACTCAAGTGATCCGCCTGCCTAAGCCTCCCAAAGTGCTGAGATTACAGGCATGAGCCACAGCACCTGGCCTTAAATTCTATTTAAAAATATAATATTTTTGGCCGGGCGCCGTGGCTCACCGCCTGTAATCCCAGCGCTTTGGGAGGCCGAGGCGGGCAGATAACCTGAGGTCGGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCGCGTCTCTACTAAGAATACAAAATTAGCCGGGCATGGTGGCACATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCTGGGAGGCAGAGGTTGCAGAGAGCAGAGATGATGCCATTGAACGCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATATATATATATATATATATATATATATATATATATGATATTTTTCCTTCTGCCTTCTTTTATTTTCTTCATTATACATTCAATAGGACATAACATTTTCTAAAATATGAATTTGAATGTTTAGTTCATGCATTTTCCTTCTTTTTTGTTTTTTATTTTATTATTATTATACTTTAAGTTTTAGGGTACATGTGCACAATGTGCAGGTTAGTTACATATGTATACATGTGCCATGCTGGTGTGCTGCACCCATTAACTCGTCAATTAGCATTAGGTATATCTCCTAAAGCTATCCCTCCCCACTCCGCCCACCCCACAACAGTCCCCAGAGTGTGATGTTCCCCTTCCTGTGTCCATGTGTTCTCATTGTTCAATTCCCACCTATGAGTGAGAATATGCGGTGTTTGGTTTTTTGTTCTTGCGATAGTTTACTGAAAATAATGATTTCCAATTTCATCCATGTCCCTACAAAGGACATGAACTCATCATTTTTTATGGCTGCATCGTATTCCATGGTGTATATGTGCCACATTTTCTTAATCCAGTCTATCATTGTTGGACATTTGGGTTGGTTCCAAGTATTTGCTATTGTGAATAGTGCCGCAATAAACATACGTGTGCATGTGTCTTTATAGCAGCATGATTTATAGTCCTTTGGGTATATACCCAGTAATGGGATGGCTGGGTCAAATGGTATTTCTAGTTCTAGGTCCCCGAGGAATCGCCACACTGACTTCCCCAAGGGTTGAACTAGTTTACAGTTTACAGTTCCACCAACAGTGTAAAAGTGTTCCTATTTCTCCACATCCTCTCCAGAACCTGTTGTTTCCTGACTTATTAATGATTGCCATTCTAACTGGTGTGAGATGGTATCTCATTGTGGTTTTGATTTGCATTTCTCTGATGGCCAGTGATGGTGAGCATTTTTTCATGTGTTTTTTGGCTGCATAAATGTCTTCTTTTGAGAAGTGTCTGTTCATGTCCTTCGCCCACTTTTTGATGGGGTTGTTTGTTTTTTTCTTATAAATTTGTTTGAGTTCATTGTAGATTCTGGATATTAGCCCTTTGTCAGATGAGTAGGTTGCGAAAATTTTCTCCCATTTTGTAGGTTGCCTGTTAACTCTGACGGTAGTTTCTTTTGCTGTGCAGAAGCTCTTTAGTTTAATTAGATCCCATTTGTCAATTTTGGCTTTTGTTGCCATTGCTTTTGGTGTTTTAGACATGAAGTCCTTGCCCATGCCTATGTCCTGAATGGTAATGCCTAGGTTTTCTTCTAGGATTTTTATGGTTTTAGGTCTAACGTTTAAGTCTTTAATCCATCTTGAATTAATTTTTGTATAAGGTGTAAGGAAGGGATCCAGTTTCAGCTTTCTACATATGGCTAGCCAGTTTTCCCAGCACCATTTATTAAATAGAGAATCCTTTCCCCATTGCTTGTTTTTGTCAGGTTTGTCAAAGATCAGATAGTTGTAGATATGCGGCGTTGTTTCTGAGGGCTCTGTTCTGTTCCATTGATCTATATCTCTGTTTTGGTACCAGTACCATGCTGTTTTGGTTACTGTAGCCTTGTAGTATAGTTTGAAGTCAGGTAGCGTGATGCCTCCAGCTTTGTTCTTTTGGCTTAGGATTGACTTGGTGATGTGGGCTCTTTTTTGGTTCCATATGAACTTTAAAGTAGTTTTTTCCTATTCTGTGAAGAAAGTCATTGGTAGCTTGATGGGGATGGCATTGAATCTATAACTTACCTTGGGCAGTATGGCCATTTTCACGATACTGATTCTTCCTATCCATGAGCATGGAATGTTCTTCCATTTGTTTGTATCCTCTTTTATTTCATTGAGCAGCGGTTTGTAGTTCTCCTTGAAGAGGTCCTTCACGTCCCTTGTAAGTTGGATTCCTAGGTATTTTATTCTCTTTGAAGCAATTGTGAATGGGAGTTCACTCATGATTTGGCTCTCTGTTTGTCTGTTGTTGGTGTATAAGAATGCTTGTGATTTTTGTACATTGATTTTGTATCCTGAAGACTTTGCTGAAGTTGCTTATCAGCTTAAGGAGATTTTGGGCTGAGACAATGGGGTTTTCTAGATATACAATCATGTCATCTGCAAACAGGGACAATTTGACTTCCTCTTTTCCTAATTGAATACCCTTTATTTCCTTCTCCTGCCTAATTGCCCTGGCCAGAACTTCCAACACTATGTTGAATAGGAGTGGTGAGAGAGGGCATCCCTGTCTTGTGCCAGTTTTCAAAGGGAATGCTTCCAGTTTTTGCCCATTCAATATGATATTGGCTGTGGGTTTGTCAGAGATAGCTCTTATTATTTTGAGATACGTCCCATCAATACCTAATTTATTGAGAGTTTTTAGCATGAAGGGTTGTTGAATTTTGTCAAAGGCCTTTTCTGCATCTATTGAGATAATCATGTGGTTTTTGTCTTTGGTTCTGTTTATATGCTGGATTACATTTATTGATTTGCATATATTGAACCAGCCTTGCATCCCAGGGATGAAGCCCACTTGATCATGGTGGATAAGCTTTTTGATGTGCTGCTGGACTCAGTTTGTCAGTATTTTATTGAGGATTTTTGCATCCAAGTTCATCAAAGATATTGGTCTAAAATTCTCTTTTTTGGTTGTGTCTCTGCCTGGCTTTGGTATCAGGATGCTGCTGGCCTCATAAAATGAGTTAGGGAGGAGTCCCTCTTTTTCTATTGATTGGAATAGTTCAGAAGGAATGGTACCAGTTCCTCCTTGTACCTCTGGTAGAATTTGGCTGTGAATCCATCTGGTCCTGGACTCTTTTTGGTTGGTAAGCTATTGCCACAGTTTCAGAGCCTGTTATTGGTCTATTCAGAGAGTCAACTTCTTCCTGGTTTAGTCTTGGGAGGGTGTATGTGTCGAGGAATTTATCCATTTCTTCTAGATTTTCTAGTTTATTTGCATAGGGGTGTTTGTAGTATTCTCTGATGGTAGTTTGTACTTCTGTGGGATCGGTGGTGATATCCCCTTTATCATTTTTTATTGCTTCTATTTGATTATTCTCTCTTTTCTTCTTTATTAGTCTTGCTAGGGATCTATCAGTTTTGTTGATCCTTTCAAAAAACCAGCTCCTGGATTCATTAATTTTTTGAAGGGTGTTTTGTGTCTCTATTTCCTTCAGTTCTGCTCTGATTTTAGTTATTTCTTGCCTTCTGCTAGCTTTTGAATGTGTTTGCTCTTGCTTTTCTAGTTGTTTTAATTGTGATGTTAGGGTGTCAATTTTGGATCTTTCCTGCTTTCTCTTGTGGGCATTTAGTGCTATAAATTTCCCTCTACACACTGCTTTGAATGTGTCCCAGAGATTCTGGTGTGTTGTGTCTGCTCTCGTTGGTTTCAAAGAACATCTTTATTTCTGCCTTCATTTCGTTATGTACCCAGTAGTCATTCAGGAGCAGGTTGTTCAGTTTCCACGTAGTTGAGCGGTTTTGAGTGAGTTTCTTAATCCTGAGTTCTAGTTTGATTGCACTGTGGTCCGAGAGACAGTTTGTTATAATTTCTGATCTTTTACATTTGCTGAGGAGAGCTTTACTTCCAACTATGTGGTCAATTTTGGAATAGGTGTGGTGTGGTGCTGAAAAAAATGTATATTCTGTTGATTTGGGGTGGAGAGTTCTGTAGATGTCTATTAGGTCCGCTTGGTGCAGAGCTGAGTTCAATTCCTGGGTATCCTTGTTAACTTTCTGTCTCGTTGATCTGTCTAAGGTTGACAGTGGGGTGTTAAAGTCTCCCATTATTATTGTGTGGGAGTCTAAGTCTCTTTGTAGGTCACTCAGGACTTGCTTTATGAATCTGGGTGCTCCTGTATTGGGTGCATATATATTTAGGATAGTTAGCTCTTCTTGTTCAATTGATCCCTTTACCATTATGTAATGGCCTTCTTTGTCTGTTTTGATCTTTGTTGGTTTAAAGTCTGTTTTATCAGAGACTAGGATTGCAACCCCTGCCTTTTTTTGTTTTCCATTTGCTTGGTAGATCTTCCTCCATCCTTTTATTTTGAGCCTATGTGTGTCTCTGCACATGAGATGGGTTTTTTCCTGAATACAGCACACTGATGGGTCTTGACTCTTTATCCAATTTGCCAGTCTGTGTCTTTTAATTGGAGCATTTAGTCCATTTACATTTAAAGTTAATATTGTTATGTGTGAATTTGATCCTGTCATTATGATGTTAGCTGGTTATTTTGCTCATTAGTTGATGCAGTTTCTTCCTAGCCTCAATGGTCTTTACAATTTGGCATGATTTTGCAATGGCTGGTATCGGTTGTTCTTTTCCATGTTTAGTGCTTCCTTCAGGAGCTCTTTTAGGGCAGGCCTGGTGGCGACAAAATCTCTCAGCATTTGCTTGTCTGTAAAGGATTTTATTTCTCCTTCACTTATGAAGCTTAGTTTGTCTGGATATGAAATTCTGGGTTGAAAATTCTTTTCTTTAAGAATGTTGAATATTGGCCCCCACTCTCTTCTGGCTTGTAGAGTTTCTGCCGAGAGGTCTGCTGTTAGTCTGATGGGCTTCCCTTTGTGGGTAACCCGACCTTTCTCTCTGGCTGCGCTTAACATTTTTTCCTTCATTTCAACTTTGGTAAATCTGACAATTATGTGTCTTGGAGTTGCTCTTCTCAAGGAGTATCTTTGTGGCGTTCTCTGTATTTCCTGAATCTGAATGTTGGCCTGCCTTTCTAGATTGGGGAAGTTCTCCTGGATAATATCCTGCAGAGTGTTTTCCAACTTGGTTCCATTCTCCCTGTCACTTTCAGGTACACCAATCAGACGTAGATTTGGTGTTTTCACATAGTCCCATATTTCTTGGAGGCTTTGTTCGTTTCTTTTTATTCTTTTTTCTCTAAACTTCCCTTCTCGCTTCATTTCATTCATTTCATCTTCCATCACTGATACCCTTTCTTCCAGTTGATCACATCAGCTCCTGAGGCTTCTGAATTCTTCACGTAGTTCTCGAGCCTTGGCTTTCAGCTCCATCAGCTCCTTTAAGCACTTCTCTGTATTGGTTATTCTAGTTATACTTTCGTCTAAATTTTTTTCAAAGTTTTCAACTTCTTTGCCTTTGGTTTGAATTTCCTCCTGTAGCTCGGAGTAGTTTGATCGTCTGAAGCCTTCTTCTCTCAACTCGTCAAAGTCATTCTCCGTCCTGCTTTGTTCCGTTGCTGGTGAGGAGCTGCGTTCCTTTGGAGGAGGAGAGGTGCTCTGCCTTTTAGAGTTTCCAGTTTTTCTGCTCTGTTTTTTCCCCATCTTTGTGGTTTTACCTACTTTTGGTCTTTGATGATGGTGATGTACAGATGGGTTTTTGGTGTGGGTGTCCTTTCTGTTTGTTAGTTTTCCTTCTAACAGACAGGACCCTCAGCTGCAGGTCTGTTGGAGTTTGCTAGAGGTCCACTCCAGACCCTGTTTGCCTGGGTACCAGCAGCGGTGGCTGCAGAACAGCGGATTTTTGTGAACCGCGAATGCTGCTGTCTGATCATTTCTCTGGAAGTTTTGTCTCAGAGGAGTACCCGGCCGTGTGAGGTGTCAGTCTGCCCCTACTGGGGGGTGCCTCCCAGTTAGGCTGCTCGGGGGTCAGGGGTCAGGGACCCACTTGAGGAGGCAGTCTGCCTGTTCTCAGATCTCCAGCTGGGTGCTGGGAGAACCACTGCTCTCTTCAAAGCTGTCAGACAGGGACATCTAAGTCTGCAGAGGTTACTGCTGTCTTTTTGTTTGTCTGTGCCCTGCCCCCAGAGGTGGAGCCTACAGAGGCAGGCAGGCCTCCTTGAGCTGTGGTGGGCTCCACCCAGTTGGAGCTTCTCGGCTGCTTTGTTTACCTAAGCAAGCCTGGGCAATGGTGGGTGCCCCTCCCCCAGCCTCCCTGCTGCCTTGCAGTTTGATCTCAGACTGCTGTGCTAGCAATCAGCGAGACTCCGTGGGTGTAGGACCCTCCGAGCCAGGTGCGGGATATAATCTCCTGGTGCGCCATTTTTTAAGCCCGTCGGAAAAGCGCAGTATTGGGGTGGGAGTGACCCGATTTTCCAGGTGCCGTCTCTCACCCCTTTCTTTGACTAGGAAAGGGAACTCCCTGACCCCTTGTGCTTCCCGAGTGAGGCAATGCCTCACCCTGCTTCGGCTCATGCACGGTGCGCTGCACCCACTGTCCTGCATCCACTGTCTGGCACTCCCTAGTGAGATGAACCCGTTACCTCAGATGGAAATGCAGAAATCACCCATCTTCTGCGTCGCTCACGCTGGGAGCTGTAGACTGGAGCTGTTCCTATTCGGCCATCTTGGCTCCAGCCTGACTGCATTTTCCTTCTTGTTGTAAATAATAAAAATATTTATAGGGACAATGCTTAGGGGTACAGCTTTGTCTCTGTCTTGTAGGTTTTAATATGTGGCATTTCAATTTTTAATATTTTCTAAATGTCTTTTGTATTCTTATTTTCCCTTCCTCTTTGGTTCAGTTTCTTGCAGATTCTTCTTTAAGGATGTTTATAGTAACATTTGTGGTATCTGACTTGGCAAGTTGCAAGTATTGTACTACATTTCCCACATTAGTTACCCTTGTAGCTGTACTTTTCCTTTCAGATCCTCTAGTAATTCAAAGAGACCCACTCAGGTTCAGGCAAATATAAGAAGCTTCTTGTGAATGGAATGGCTTATCTAATATTTCTAAAATGACTGGAGCAATGACTTACTGTTTCTAAGGCTTGAGGGACTTGGGCGCAGAGGCAAAAAATCACTCCCGCTAGAGGTATGATGGTGAAAGCTGGCTAGCAGCTGTTTGTACCTCTGGTGGGGAGTAGGACTGGGGACTCCCAGACCTTGCAGGCACTTATTAGGGAATACAACATCATCAGGAAAGGGACTATGTGAACAAGGTTGGATGCTCCAAGGAAAAATGGATTGATGACCCTGGGAGGCTTTCCTCCTGGAATCTGGGCAATGCCCACTCTCGTCATGCTTCTTTAGAACATGTTCTTATTTTCAAATGTTTTCATAGATTTTAAATGGGAATTTGGGAAGGCAGTCTCCAGTCACTTCCACCCTACCTTTTTCCTGGAAGTGTTCTCTAAATCATGTACACATCTATTCTCCTTTACTTGGCTCTAGTGGTTTCTGGGACACCGGTTAGTGGTCAGACTTTGAAAACTGCCCTTTCTTTGACTACAAGGTCTGGTTTGGTCTATATGTCTCTGGAGAATTCTTCTCCCTTCTAGGTGGTGTCTTTGTAGGGACTGTCTGTCTCTGTTGCTGGGTGGAACCTGGAACCTGGTTAAGGCTGGCCAGTGGAGGGGCTGTGCCTTGAGGAGGGCACAGATAGCCCCAGGAATAGGTGCCATCCTGGCAATGTCCTTGGGGAATGTGGTCTCCCCTTTGGAGATTTGGCTGTTGCATGAGGTGCTAACTGAGTGCAGGGGTTGCTACTCGTTGCTTACTGCAGCTTCCCTGGAAGCCTTTGATGAGGAGGGCAGGACCTATGGAGGAGACAGATCAGGAGAGTGCATTGTTTCCACAGCTGCGGTTTGCTCTGCCTAGCCCTTGTGGTGAGATGCAAGGAGCCTTCTTTCTCTATTATGGGAAGGTAATTTGCTCTGAGGCCATTTTCACTATTAAAATACCCATAAGGCAGGGAGGAAAACCCCTCTTTAAGCAGAGAGGCAGGAGTCAAGTGTGGGGAAAGCTGTTGCTCTCCCCTGGAAGTTTTGGATGAACAGTGGATACGTGAATGTGGGGTGGGAGGAGGCTGTTAAAAATGTCTTCATACAAGGAGGCATGTAATTTCCTCCTGGTGGGCATGGTTTTACTCCACATCAGATGTCATTTTGATCTGCCTGTCCCATCAATGGTCAGGCTTCAGCTGGAAAAGAGAGAATGGTGGAAGTAAGAGGTGGGAGACTAGAATGTCAGGCAGAAACAGCAGCGACAGTCTGCTGTCTCATTTGCTTAAGATCTGTATGAAGACTTGAAAAATCCCACAAATCTGCAATTGCAGATTTTTATCAAAGCATCTATAACTTTATAATTTAAAACTGGTATTAAAAGATGTTGCCTTTTTAAACTCTTTTAATGCCTGTTTATATGAGGACATTTTTAACAACCTCCTGCAACCCTACATTCACACACACATATTTTAGGACAGACATTATTTAATCTGATATGCTCATTTGTCACACAAAAGGTGAGCAGCTCAGAAAAAGCCTGAAGCAAGGAGGAGCTCTTGCCTGTAGCAGCCTCATGACCATTTCTGCTCTTGGACTTGGCTGCTTTGAGTTTTTTCAAGTCCTCAAGGCCTTTGTTCTTTAGCTTTCAGGGCTATTATGACTGGATAGTTTACCAGAAAAGGTCCACTTCTTTTACTCTGAACTGATAAGCTGTTTAACTGAATTGCTTGTTTCTGTCCCAAGTGAGCGTGTGTATGTGTTTGTGCTTGCAGACTCAGTATTTTTGCAGCTTGGGATTATAACAAAAGATACGAGATTTTTTTTTTGAGACAGCAGATAAAAAGGGAAATAAATTAGAGAATTATCTTAGTCAATTTTATGTTGTGATAACAAAATACCACAGACTGGATAATTTGTAAAGAATACAGATGTATTTCTTACAGTTCTGGAGACTGGGAAGTCCAAGATCAAAGGGTCTGTATCTGGTGAGGGTCTTCTTGCTGCATCATCCCATGGCAGAAGGCAGAAGGGCAAAAATGTGCACATGAGAGAGCAAGGGTAAAGAGGCCTGAACTCACCTTTTATCAGGAACCCACTCCTGAGATATTGGCATTAATACATCACAAAGGAAGTGTCTTCATGGCTTAATCACCTCCTAAGGGTCCCACCTCTTAATACCATAATAATGCCAATTAAATTTCAACATGACTTTTGGAGGGAACATTCAAACCATAGCAAGGATAGAATATGATGGAAGATTCAGGGAAGAGAAAGGAAGGGCCCAGTTTTTATTAAAATTGTGACAAAGTACACATAACATAAAAGATACCGTCTTAACTATTTTAAGTTTATTGGAATTAAGTGCATTCACAGTGCTGCACAACATTATCATTATTCATCTCCAGAACTCTTTTCATCTTGCAAAACTGAAAGTCTATATCCATGAAATACCAACTCCCCATTTCAATCTTCCCCTCAGCCCCTGCCTCATATAAGTGGATCACACAGTATTTGTCTTTTTATGACTGGATTGTTTCACCTTGCATAATGATTTCAAGGTTTATACATGTTGTAGAATGTGGCAAAATTTTCTTCCTTTTTCAGGCTGATTAATATTCCATCACATGTATATACCACATTTTACTCTGAACTGACAGGTTGTTTGACTGAATTACTTGTTTCAGTCCCAAACATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCACAGACCCAGTATTTTTGACAGTGTGGGAATATAAAAAAGATGTAAGCTATTTTCTTTTTTTAGGAGATTGTCTACTTATCTGGCAAAGGGCATTTGGGTTGCTTCCACCTCTTTGTTATTATGAACAGTGCTGCTATGATCATGGGTAGGCAAATAAGTTCTTGAGAACCTGCTTTCAATTCTTTTATACATATACCTAGAAGTGGCTTTGCTGGATCATATAGCAGGTCTATTTTTAATTTTTGAGGAGTCTTCATACTGTTTTCCATAGGGATTGCACCATTTTACAATCTCATCAACAGTGCACAAGTGTTCCCATTTCTTCACATCTTCATCAACACTTGTTATTTTAGGTTTTTTAAAAAATAGTAGCCATCCTAATGGGCATGAGGTCATCTCTTTGTGGTTTTGGTTTGCATTTTTCTAATGGTTAGTGATGTTGAGCATCTTTTCATATGCTCGTTGGCCATTTGTATTTCATCTTTGGAGAAATATCTATTCAAATCCTTTGGTCATTTTTTAATTAGGTCATTTAATTTTTTATTGTTGAGTTGTAGGAGTTCTATTTTATTTTATTATTTAATTTAATTTTATTTTACTGTAAGTTCTGGGATACAGGTGCAGAACATGAAGGTTTGTTACATAGCTGTATGTGTGCCATGGTGGTTTGCTTCTCCATCAACCTGTTATCTAGGTTTTAAGCCCCACATGCATTAGGTATTTGTCCTAATCCTCTCCCTCCCCTTGCTCCCCACCCCCAACAGGCCCCAGTGTGTGTTGTTCCCCTCCCTGTGTCCATGTGTTCTTATTGTTCAACTCACACTTAGAGTGAGAACATGCAGTGTTTGGTTTTCTGTTGTCAGAGTTATTTACATATTCTGGATGTTAACCCCTTGTTAGGTATATGACTTTCAAATTTTTCTTCTATGAGTTTTATGTTTTTCAGGTCTTACATTTAGGACTTTAATCCATATTGAGTTAATTTTTTATATGGTGTAAGTAAAGATTCTAACTTCATTTTTTGCAAGTGGAGATTCAGTTTTCCCTTTACCATTTGTTGAAAAGACTGTCCTTTTTCCAAGGACATTGCCAAGACTATTCCATAGTCTTGTCACCCTTGTGGAAAATAATTTGGCCATATATGCAAAAGTTTACTTCTGGGCTCTCTATTCTATTCCGTTGGTCTATATGTGTCTTTATGCTAGTACCACCTTGATATGGTTTGTCTGTGTCCCCACCCAAATCTCATCATGAATTGTAGTACCCATAATCCTAACGTGTGGTAGAGGGCCCTGGTGGGAGATAATTGAGTCACGGCGAGGGTTTCCCCCATGCTATTCTCGTAATAGTGAGTTCTCATGAGATCTGATAGTTTCATTAGGGGCTTTCCCCTTCGCTTCACTCTCATTCTTCTCCTTCCTGCTGCCGTATGAAGAAGGACATGTTTGCTTCCCCTTCCACCATAATGGTAATTTTTCTGAGGCCTCCCCAGCCATGCTGAACTGAACTGTGAGTCAATTAAATCTCTTTCCATGATAAATTACCCTATTTTGATTACTGTAGCTTTGTAATAAATTTCGATATCAGGAAGTGTGAGACCTCACAATTTGTTTTTTTTCAAGATTGTTCTGGCTATTTGGGGTCACTTGAAATTCCATATGAATTTTAGAATGGATTTTTATATTTTTGTAAAAAATGTCATTGGGAGTTTGATAAGGATTGCACTGAATCTGTAGATCACCCTGGGTAGTACGATATCTTAACAATATTAAACCTTCCAATCCATGAACACAGGTATGAAAAAATTCAGCTATTTGCTTGTCATTTACTCTCTTCATTGTTCTGTCACCATTGTCTCCTTCTGTACATCAGAGTGACAGCTCTAGAATGGTGGTGACAAAGTACAACCTCCAGTGCTTGGGTCTCATGTTACCTGAAACTATTCTCAAGAATATGGACATAAAGCTAGGCCTTGAAATGCAGCTTCACAATTTATCTCTGGGCCTGTTTCTCTGAAATTCTAAATACCAGGACTTGCTTAAAAAGCTTGCATTTTTGAACTCTGGTTTGCTGAAGACCAGAATGTTAAATGTCACATTGAAATGGAAAAAAAAATCAAGGCCAGTAAGATAGAAGGGCTCTTTGCTTTTCACATTGTTTATCATGTTTCTTCCTGTTGTAAATGATTTGCTTTTTAGGTGTGTCTTCACTGGGATTTATATTTTTGAAGCTTTGATTAAAATATTGGCAAGAGGTTTCATTCTGGATGAGTTTTCTTTCCTTCGAGATCCATGGAACTGGCTGGACTCCATTGTCATTGGAATAGCGTAAGAATATTAAAGATGTTTTTGAAGAGACTTGGAGTGGGAAAGAAGCCCCTTTGCTTCCATCAGAGCTCTGTGTGGATCCATTTGAGTGGCTGAGTATGGCCTAGTAATGCCTAGAGCTCTGAGTGCTGTGTTGGTTGGCCCTGAACAGCACGTGCAGCCAATTGGCCAGAGGCCCCCACACACAAAGCCATTTCTAGGTCAACACTCAAGTTCGTGTTCCTTCAGCCATTTACTCACCAACACTGGCCTGTTATGGGCCAGGCTCTATGGAAGATACAGGGGTGTGCCAATGAAATTAAAAAAAAAAAAAAAGACATGGTTTTCAATCTTATGAAGCTCACAGTCCAGAGGGGAAAAGTAACAATTAATGGCCCTGAAAATATGGTATGATGAGTACCAGGATAGGGGCTATACATGTATTAAGCTGTCAGAGAATATAGGAAGAACAAAGCAGTCTAAAAAATCTTTGGCTGCTACAAGGTTACAAAGAGCTTTACTCCTGTTTACTTCTAGAAGTTCTGTAGTTTTGGCTTTTATGTTTCAGTCCATGGTCCATTTTGAGTTAATTATCATGTATGGTGTAAGGTGAAGGTTGAAGCTCTTCCTGAAGGAAAGACACTGATTTGGAATCTGAATGATGAAATGGAGTCAGCAAAAGAAAAAATAAGAAATGCAGTGACACATTGCCAGCACAACAATAGTAAACCTGGCATCATTCAATATGTACCAGGCATTATGTTAAGCACTTTGCATATTTTTTCCTATTTAATATTCACAAAAGCCCTTGGAGGTAGGTGCTAGTAATTTCAGAGTTTTACATTTGAAGTTCAAAGATTGTGTGCTCTTCGTTATTGAAAGGTGGTGAAGCACAGTGGTTTAGAGTATGGCTCTGGAGCTACACTGCCTGGCTACACATTCAGGCTTGACCATGTAGTGGCAGGAGACTTTAGTGAAGTGCCTTGGGGCATGGAACTTGTCAGCCACTCAGTAAATATTTGAGAGGACTTCTATTATTCATGGATGTAGCCACAACTACATGAACTATTTCAAGGAGTCTTCATCTGTGTCCACGTATGAAGGTGAACTGAGAACAGCGTCCTCTGCAGTTCATAGCAAGAACAGTGGTATTGCCAGATCCTAAGATAAAGGTGGGGCTGGAAAGGTGAGGCTGTCGATTTCACCTTGGAGGTCACAGGCCTCAGGCTGAGCTTACTTGTGGCTTTTGTGGAGATGTGCAGTGCAGCTGCAGGACTGAGCAAGCCCATGCCTTCCCTGTGCTTTGTCTTGTAGGATTGTGTCATATATTCCAGGAATCACCATCAAACTATTGCCCCTGCGTACCTTCCGTGTGTTCAGAGCTTTGAAAGCAATTTCAGTAGTTTCACGTAAGTCACTCTCACTTTCCACACTGGCTCCTATCTAGAAAGAATGTTAATTTATATTTAAATTTTTTCAAAATGTCATCATGTAAACCAAAAACAAACAAAGCCTGCTTTACAAATTTATTTGATGGTCCAACTGATAATTATTGAGCACCTAATATGTCCAGGCACTGTGCTAACTGCTTGAGATCTAGTAGTAAGCAAATGGATACGGACTCATGGAGTTTTACCTTCTATGGTGGGGGTGGGGAGACAGACACGAAGCAACAACTATAATAAATAAGTACATTATGTAGCATGTGAGCAAGTGCTATGTGCCAGGGAAAATATAAAGAAAAAGTAGACACCATGAGGGCTATGGGAAGTCACAGGGTACAGGGGAAGGAGGTTGCAGTTTTAAACAGAGTGATCACGATAGTTGTCATTGAGGTGAGAGGTGGCAGAGATTTGAAGGTGGCAAGGGAGTGACACAAGCAGATATATGGGAAAGAGTATTCTGAGCATGGGAAACAGCCAGCACAGAGATTGAGGAGTAGCAAGGATGAGGATGGGACTGGAAAGGAGAGGGTGAGAGGCCAGGTAGAGGTGGGATCATAGAAGTGAAGGGGGGGCCGGGTACAGTTGCTCACGCCAGTAATTCTAGCACTTTGGGAGGCCAAGGTGGGGAGATAACTTGAGGCCAGGAGTTTCAGAACAGCCTGGCCAATATGGAAAAGCCTCGTCTCTACAAAAATACAAGAGTTAAGGCCAGGTGCAGTGGCTCACTCCTGTAATCCCAACACTTTGGAAGGCTGAGGCAGGTGGATCACCTGAGGTCAGAAGTTTGGTATCAGCCTGATCAACATGGAGAAACCCCATCTCTACTAAAAATACAAAAGTAATCAGGTGTGGTGGTGCATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGTGGGAGAATCACTTGAACCTGGGAGAGGGAGGTTGCGGTGAGCCTGGATCGCCCCATTGCACTCCAGCCTGGGCAACAAAAGCAAAACTGTCTCAGAAAAAAAAAAAATTAGTTAGATGTGGTGGTGAGTACCTTTAATCTCAGCTACTCGAGAAGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCAGAGGCTGCAGGCTGCAGTGAGCCAAGATTGTGCCATTGCACTCCAGCCTAAGGTCTAAAAAAAAAAAAGGACTGAAGGGGAGTCTTGTAGGCACAGGGAGGATATGCGCTTCACTCTGAAGAAATGGGAGCTGTTGTGTGGAGAATGGAATGTAGGGGAGATGGGAGAAGCAGGAAGACCTCCTGCTACAGGGAGCACAGTGGACTAAAGATCCCGGCTTCCACCTCTCTGGATTCCCCACTGCATTCATCCACATGCCAAGATCATGCCTGCCCTGCACAGTGTGTGTGGGTTCTAGTGCTGGCCCATTCCTGAAGCATATGGGATTCTTCTAATAGGAAACTTTGGCTCAGGGACTCTCCAGTGACCTGGCTGAGACTCCGACAGCTGCACTGCAGTCTGAGGTTCTTCCTATCCAATCCTCCTCCCTTGTCTGTCTCTCAGACATCAAACTTGAGCTGCAGTCTGAAGTTTCTCCCTGCCCACTCCTGCTCCCCCGACTCATCTTTCACAGGCGTCCTCCCAACCCCCACAAATCTCTGGCACATCTGATCTCAGAATGTCTGCTCCTCAAAACCCAAACTGACACAAGAGACCATAATCCAAGCAAGGGATGGCAGTGCTCTCTGATCAGGTAGTGGTAGTGGAGTTGGTAGACACTGGTTTGATTCTGTGTCAATTCAAAGGTAGAAACAATAGCATTTCCTGACTGATCAATTGTATACATGGTGTGAGCAAAAGAGAGGAGTCAAAAATGACTTTTATACCTGATGTCACTGATTGAGACAGAGAAGGCTGTGAGAGGAGCAGGCCTTGGGGTCAGATTACAAGCTAGCTTTGAACATGTTATGTTTGAGATGGCTGTGAGACCACCAAGGGAAAGGATTCAGCAGGAAATTAGGTATATATGAGTCCAGGAGTCCAGGGAGAAGTCAGAGCTAGAGATACCATTTCAGGAATCAAGGGTCTATAGATATTTAAAGCCGTGAGATCTAAAGAGATCACCAAGGGAATAAGCACTGATGGAGAATAGAAGAGGACCAAGGTCTGAGCCCCAGGGTACTGAAACATGAAAGGGAGAAGAGGAGAAACCAGGGAAGGAGATGAGCAGCAGCCACCTGTGAAGTAGGAGGGGAACCAAAGGTGGTCTCCTGGAAGCCAGGTGAGGAAGTCATGTTAGCCCTTCGGATCAGGATGAAGCCAAGGTTGGGTGTGAATATGAATTTCTGTTTATTAAAAATGAGGTTATTATAGCACACCCTTTTCCTCTCCTTCCCCTTTCCTCTCTTTTCTTTACTAAAACAAACAAACAAAACAGTTTATTATCTTCCCTCTCTGGACTCCTTTCCCCCCACCTTCTCCCCTTCCCTTCTTTTCTCTTCTATCAGTGGCTTTGGCAAACTGTAAAAAGACATGGGAACTATTTAGTTGTGCTGATGATATAGACATAAACTATACCGCATGGCCCCAATACCTAAACTCCAGTCCTTTCAAACAGAAGCAGTAGGTTATTTTTTGAAACTTCAGAAAAGCAACTGAATAGTGATAAAGAAACTGGTAGTCAGAGGACACTTTATATACAATTTATCTCTTCTTCTCCTCCTCCTCCTCCTCCTCCTTCTCCTTCTTCTTTTCAAGTAGGACTTTGTTTTCCCAGGTAGATTTAGGAGCTTTCTGCATGAGCTGAGCCCCTTTTATTTTCACATAAGTAAATACACATTCAGGCAGTCATTTAGCAGCTTTCATGTCCCCGTCTTCATCTCCACACAGGTGAATATAGTCTCTATTTTGGCCCATTAAATGCACTGAGATGCCTGGATGCTTTTTGGAAGTAAGGGTGTGAGGCACAATCTTTTATGAGACTATTAAAAAATGATCCAGGAATCACAATCATCACTCATCCACATTTCTTGCTTTGGAGAAGCCTCTTGTTATAGATTAAAATCTGCCTTGGGTGACACTGTAGTAACCTCTGGTTTGCCTTCAATTGGTGATATAGTAAGTGTCCCAGCTTGGCCTCCAGGTTCCTGCTATCTGGTGTTATACTTTGTGGGAAGCCCATCCACTGAGGGCTTGGCCTCCCACTGTATGCTGCCATGTGCTCTCACCTCTATGCTAATCAATTGAAATTTTGTCACTTAGTGTCATTCCATGCCTTTCAATTACATCTGGGAGTTTATCTCTCCAGCTATCTTGAGACTCAGTGGATTGCTGAAGTTGACTTGTTCTTTTTCCAGCTCTGTCCAGTGACATCATATTGGTAGCTTGAAATCTACCATGGTGGTGAGTGGGGATCAGGATCTATACCACAGAATTCAGAAAACAAGGAAACCTGTAAATGCTATAGATCAGGGCTCTCCTGCCTCCTGCTTGAAGAGCCAGTTTTAAATATTTACTAGCATGCCACAAGATTATTTATTTTGTAACTTTGGCAGCTTAACAGTGTGCCATGGACATAGTAGGTGCTTAATAAATACTTCTTGAATGAATGAATGAATGAAAAGAAAACACCCTATTCTAACTGGTAATCTGAGTTTTCCCTCCCAGGATATCCTCATAATTTGGGTCTAGACAAATCTCCTTCATCCCCTATACCCCACAATTTAGCTGTGTCTCATGGTACTCAAGTACTTTGACAGTAGACAGTATCTACTAAATGTTAAATACAGGTCTATCTCATTCTCTAGTGTGGCCCCAGCTTAGGTGGTGCCTTTGCAACTGTCCTCAACCTATGCCTTGAGTCCGTGTGGCATGTTTCTATTCAAAGCCTCTTTGCTTGCTCCTCATAGCAGCACTGAGGCAGGCATAGGGCAGGAATAGAGAGATTGAGTCTCAAACTTGTTGACTGCTTTGATGACACTCAGCTTCCAAGGCACAGAGTTGGAGCACAAACTCAAGGCATCAGAATCATAGGCTAGGCTGGAGTCCAGTTCCAGCCTGCAATTTGACAGTTTCCGAGACTTCAGTTGCTCCTTACTTGGCCTTTGTGAGTGTCTGGCTGAGGGGGATGGGCTTCTCCCCAAGCCTGTCCTCTTTCTCAGGTCTGAAGGTCATCGTGGGGGCCTTGCTACGCTCTGTGAAGAAGCTGGTCAACGTGATTATCCTCACCTTCTTTTGCCTCAGCATCTTTGCCCTGGTAGGTCAGCAGCTCTTCATGGGAAGTCTGAACCTGAAATGCATCTCGAGGGACTGTAAAAATATCAGTAACCCGGAAGCTTATGGTAAATACCCAACCTTTCTTTCACTCCTTGGTTTTTGCATGGTTAACTTTTCCTTATCCTCACTTGTCCTGCCTTCACACTTCCCTTACAGAGTCTCTTAGCAGCAAGGGAGGACTTCATCCATCCAGGGTGCTCCTGTGGGCAGATGCATTTCCTAGAGAATTGCCTCATTCTTTGGCTTCCTATGGAGAAGGAAAGTCTTGAAACCACATCCACAAGAGCAATTCTATGCTGTCCCATCCCTTTGCCATTTAGATCTCTGCCCACTTCTTTGTAATGATTTGCAGAAGTTTTTAGAATGCTTGGATTTGGCTAACAATAATCAGCAGCTTCCCAGCCCCACTCCTCAAGTATTTCTCCAATGACACGTCTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTAGCTCTGTCACCAGGCCGGAGTGCAGTGACATCACCCCACTGCAACCTCTGCCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCTCAAGTAGCTGGGATTACAGGCATGTGCCGCCACACCCAGCTAATTTTTGTATTTTTAGTAGAGACGAGGTTTCACCATGTTGGCCAGGATGATCTCCATCTCCTGACCTCGTGATCTGCCCACCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCATGCCCGGCCAACAACACTTCTTTCAGTGCCCACCCTCTCCATGCTGAGGTTGACCCTACACCTACTCCATGTGTCATGAATCTCATGTGACTACACTCTCTTGATGTCTCTCATGGGATGGGTCTGTGTCCAGCTTCCCCCAGGGAGGATCTTCGGTTCAGTATTATTTAAGAGAGGCAAAGTGGTGGTGGTGATGTCACTGCTTCCTCCTCCAACTTCTCCTTCCTTCTTCTTTTCCCTCCTTCCTTCTTACTCCTTCCAGAGGTCTTGGGCTCCAGTTTCTCTCTGGACACACCTCCTTTAATCATTTTAGAAGATGTAGAAACATCCATTTTCAAGCATAATCTGCATACTTACTATTCTCTTTCCTTTCAATGTTTTTTTTAAAATCTGAATTTATGACTTTTTCCTCTCTCAGACCATTGCTTTGAAAAGAAAGAAAATTCACCTGAATTCAAAATGTGTGGCATCTGGATGGGTAACAGGTAAGAGGTTTATAATCATTTTCCTCCCAAGAAGAGTACCTCTAGACCTTTTAGAATGGTGTGGCAAACAGTCTTTAATGACTGGTGGACACAGTGATGATAGTTCTCACATAATTATTTCATGTGTCTAAACATGTTTAGACTTGTGTTATTAAATTAAAAAATCATTTCCTGGTGTTTAAGGCAGCTTTCCTCCCTAGTTATGTTAAATTGGAAGCTTATAAGAAACCTACTGAAACAGTGATATTTCAACTTTATGGAGTAGGTATTCAAGACTTTCAAAACTGAGAAGGGATGTTATGACTGAGAGCCCTGTGAGGCCCTTGTTTCCTGTTCAGTGGCATTGCCCTGCTGCAAGAGTATCTGAGCATGCTTCTTCTCTTTGTTGACCTGTTGACAACTATTGCCTCAAATTACAAACACAGTTGAACATACATCCACTTACACTTGCCCCAACAGCTGCTGGCCCTCTGCTCCAATGGGGAAACTCTTTTTTCCTTCAGGTTAGCGGTGCTTTCTATTGTGAAAGATTCAATTCAGTGAAGATTTATTGAGTGTATTTCAAATTAAGATAATATGCAAGGTACTCTGATGAAATAGATGACATTCATTCCTAAATTACAAATAGATAACAAGAATCTACAGAGTGCCAAGCCCTGAGTTTGCCTCAAGGAATGCAATTGTGAACAAGGCAGCTCCACGTTTTTGGATTTGATGTCTTCTAGGGAAGACAGATGCACAGAAAATGCAGTTATGTGTTGCATAATGATATTTAGATTGATAATGGATGATATATATGATACTGGCCCCATAAAATTATAATACTATATTTTTACAGTGCCTTTTCTGTGTATACACAAATTATTACCATTGTGCTACAGTTGCCTACAGTATTCAGTATAGTAACATGCTGTACAAGTTTGTAGCCTAGGAACAATAGGCTATGCCACATAGTCTAGGTGTGTAGTAGGCTATACCATCTAGGCTTGTGTGTACACTCTATGATGTTTATACAACAAAATTGCCTAACTACACATTTCTTAGAATGTATTCCTGCTGTTGAGCAATGCATGATTACTAGAAAGTGTGATGAGTGTTATAAGGGTGATGTGCAGGTGCTGAGAGGTATGATGGGGTTGTGACCTTGTCTGGAGAATTAGGGAAGGCTTCTCTCATTCCATTTGCAAGACACGATAGGCTTTAATCTAATATGAAGCTTACTGAGCTTTCTAGAATGATTCCCAACAGCACACACTGCATTGATATTGTTGAGGATCATTCTAGAAAGAAAAACTGTCTTCCATGAAACCGGTCCCTGTTGCCAGAAAGCTCCCCAACATTTTGGCACCAGGGACTGATTTCATGGAAGATAATTTTTTCGTGGGCAGGAAGTGGGGGTGGTTTTAGAATGAAACTGTTCTACCTCAGATCATCAGGCATTAGTTAGATTCTCATAAGGAGCGTGCAACCTAGATCCCTTGCATGCGCAGTTCACAATAGGGTTCACACTCTTATGAAAATCTAATGCTTACCACTGATGTGACAGGAGGTGGAGATCAGGTGTAATGCTCACTCGCCTGCTGCTCACTTGCTGTACAGCCTAGTTCCTAGCAGGCCATGAACCAGTACTGCTCTGGGGCCCGGGTTTGGGGATCCCTGTCTTAAAGGGAGCCCGACAGCTGCTGCATATAGCACTTCTTTTACCCCTCACACAGGCAGACAGGTGTGGTTACCATAGGCAGAATCCAACGTGGGGTGTATAACCTAGACAGGCAGAAGCAGCAGCCTTGTTTCCAACTAGTCCTTTTTTTTTTTCCCAAGTCTTTTGCCTCCAAAATCTCTTATTACAATACTATACTGCTATAAAAAGTCCAAAAAACTATTTTAGCTGTTGAAAACTCAAATAACATCAGACACAAAATGAATCAAGTGTGATGTGTATATGACAATAGAGAGTGGTGGGGAAAGTAGAGAGGCAGAGGACACATACCATGCTTAACAGCAGTCAAACTTGGGTATTTTTTTTATTTCAGTAGGTTTTGGGGGAACAGGTGGTACTTGGTTACATGAATAAGTTCTTTAGTGGTAATTTCTGAGATTTTGGTGCACAAACTGGTCTTCTTTTTAGCTTTTATTTTAAGTTCAGGGGTACATATGCAGGTTTGTTATATAGGTAAATTTGTGTACAGATTATTTCATCACCCAGGTATTAAATCTAGTACCCATTAGTTATTTTTCCTGATTCTCTTCCTTCTCTCAACCTCCACCATCCAATAGGTCCCAGTATCTATTGTTCTCCACTGTGTGTCCATGTGTTCTCATCATTTATCTCTCATTTACAACTAAGAACATGTAGGATTTGGTTTTTCTGTTCTTGCATTAGTTTGTTAAGGTTAATAGCCTCCAGCTCCATCCATGTTCTTGCAAAGTAATAGAACTCATTCTTTTTATGGCTGCATAGTATTCCATGGCACATATGCACCACATTAGCTTTATCCAGTCTGTCATTGTTGGGCATTCAGGTTGCCTCCATGTCTTTGCTCTTGTGAATAGTGTGACAATGAACTTTCACTTGCATGTGTCTTTATGATACAATGATTTATATTCCTTTGGGTATATACCCAGTAATGGGCTTGCCAGGTCAAATGGTAGTTCTGTTTTTAGGTCTTTGAGGAATTGCCACAGTTTTCCACAATGGTTGAACTAATTTACACTCCCACCAACGGTGGATAAATGTTCCTTTTTCTCCGCATCCATGCCAGCATCTGTTATTTTTTGACTTTTTAGTAATAGCCATTCTGACTGGTGTGAGATGGTATCTCATTGTGGTTTTGATTTGTATTTCTCTAATGATCAATGATGTACAACTGGTCTTTATAGGGCTCTTGTCTCTAAGTTTATGTGCCAGCACATCTCTCTCAGTCTAGGTACAATATGAATGCTCCAGAGTTTTGTGGTAAACCAACTCCTAGTACACAGCATTCCACAGTGATCACAACATGCTTGCTTCTGAGGAGATGGCACTCTGAGAAGGCAGGTCTGTGATCATCTTTCCAGGCAGGCTTTGGTCTTTTCAGGGCCCAGGCTAGTCTTCACCCTCCAGTCACTGCTTCCTCCCCTGCTGACTAATTCACCGTGACTACAACTCAGATCTGAAGTCTAAGTAGGAGGTTCCAGTAGATAAAGAGTGAACAATTCCAGACAGTAGGCACAGCAAGGGTGAGCATAAGGCCTTGGAGCACAAGAGCATATAGGGATTGACAAGAAGCCCAGCAACTTTTTGGGCAGACAGGCAAGAGAGCAGGAAGGAACAGAGACAAGAGCCAACCATGAAGGACGTGATAGCTCATCAGTGCCAACGATTTTGGTCTTGATCACAAAGGAAGCAGGAGACATGGAAAGGTTTTAGGGCAGGCAGATGACTGGGCAGATTAGACATATTTTAAAAGCCACTATGGTTTCTGTATAGAGAATGGATGCATAGAGTAGATGAGGAAGCCAATTACAAGACTGTGGTAGAAAGAATAAGGATCATGAAGATGTACAGTGGTGATGAAGAAAGCTGGATGGATGCATTCCAGAGACGTTTAGGAGACAGAAAGACTAGGACATATCAATATTAGCCTTGAATGTCAATGGGCTAAATGCCCCACTTAAAAGGCACACAATGGCAAGCTGGATAAAAAAGCAAGACCCAATGGTATGCTGTCTTCAAAAGACTCATCTTACATGTAATGACACACATAGGCTCAAAATAAAAGAAATGGAGGAAAATATACCAAGCAAATGGAAATCAGAAAAAAAGCAGAAGTTGCAATCTTAATTTCAGACAAAACAGACTTTAAACCAACAAAGATAAAAAATAGATAAAGAAGGGCATTACATAATGGTAAAATGTTCAATTCAACAAGAAGACCTAACTAATCTAAAGGTATATGCGCCCAAGACAGGAGCACCCAGATTCAAAAGGCAAGTTCTTAGAGACCTACAAAGAGACTTAGACTCCCACACAATAATAGTGGGAGACTTCAACACAGCACTGACAGTATTAGACAGATCACTGGGGCAGAAAATTAACCAAGATATTCAGGACCTGAAGTCAACATTGGACCAAATGGACCTGTTAGACCTCTACAGAACTCTCCACCCCAAAACAACAGAATATACATTCTTCTCATCACCACATGGCCCATACTCTAAAACTGACCACATAATGGAACATAAAACAAGCTTCAGCAAATGCAAAAGAACCAAAATTTTACCAAACACACTCTCGGACCACAGCACAATAAAAATAGAAGAAAAGACTAAAAAAATCACTTAAAACCATGCAATTACATGGAAATTAAACAACATGCCTCTGAATGACTTTTGGGTAAATAATGAAATTAAAGCAGAAATCAAGAAGTTTTTTGGAACTAATGAGAACAAAGATACAACATACCAGAATCTCTGGGACACAGCTAAGGCAGTGTTAAGAGAAAATTCATAGCACTAAACGCCCACATCAAAAACTTAGAAATAACTTAAACAATGTCACATCACAATGGAAAGAGTTAGAGAAGCAAGAGCAAAGCTAGCAGAAGACAAAAAATAAGGAAATCCAAATAAACACAATTAGAAATAACAAAGGGGATGTTACCACTGACCCCACAGAAATAAAAATAACCATTAAAAACTGCTAAAAACACCTCTAAGCACACAAACTAAAAATACCTGGACACAAACACCCTGCCAAGACTGAACTGGGAAGAAACTGATTCCCTGAATAGACCAATAACAAGCTCCAAAATTGAATCTATAATAAATAGCCTACCAACCAAAAAAATCCTGAGACCAGATGGATCCATAGCTGAATTCTACTAGATGTACAAAGAGCAGGTACCATTCCTACAGAAACTATTCCAAAAAATTGAGGAGCAGAGACTCCTCCCCAAATCATTCAATGAGACCACCATCGTCCTGATACCAAAACCTGGCAGAGGCACAAAAAAAGAAAACTTTAGGTCAATATCCTTGATGAACATTGATGCAAAAATCCTCAACAAAATACTTGCAAACCTAATCCAGCAGCACATTAAAAAGCTAATCCACCACAATCAAGTAGGCTTCATCCTTGGGATGCATGGTTGGTTCAACATATGCAAATCAATAAATGTGATTCATCAGAACTAAAGTAAAAAAACACATGAATATTTCAATAGTTGCAGAAAAGGCTTTTGATAAACTCAACATCCATTCATGTTCAAAACTCTCAATACACTAGATATTGAAGGAACATACCTCAAAATAATAAGAGCCATCTATGACAAACCTACAGCCAACATCATACTGAATGGGGAAAAGCTGGAAGCATTCTCCTTGAAAAGTAGCACAAGACAGGATACCCTCTCTCACCACTCCTATTCAACATAGTATTGGGAGTCCTGGACAGAGCAATCAGGAAGAAAAAGAAATAAAGGGCATCCAAATAGGAAGAGAAGAAGTCAAACTATTCCTGTTTGCAGATAACATGAATCTATATCTAGAAAACCCCATAATCTCAGCTCAAAAGCTCCTTCAGCTGATAAACAACTTCAGCAAAGTTTCAAGACACAAAATCAATGTACGAAAACCACTAGCATTCCTGTATACCAACAGCCAAGCCGAGAGCCAAATCAGGAATGCAATCCTATTTACTATTGTGACAAAATAATAAAATACATAGGAATACAGCCAACAGGGAGGTGAAAGATCTCTACAATGAGAATTGCAAAATGCTGCTCAAAGAAATTAAAAATGACACAAACAAATGGAAGAACATCTCATGCTCATGGATAGGAAGAATCAATATCATTAAAATGGATATACTGCCTAGAGCAATTTACAGATTCAGTGCTATTCCTATGAAATTACCAATGACATTCTTGACAGAATTTAAAAAATGCTGTTTTAAAATTTATATGGAATCAAGAAAGAGCCCAAATACCCAAGGCAATCCTAAGTAAAAAGTATAAAGCTGGAGGCATCACGTTTCCCAACTTCAAAATATACAACAGGGCTACAGTAACCAAAACAGCATGGTACTGATACAAAAACAGTCATATAGATCAATGTACAGAATAGAGAGCCCAGAAATAAGGCTGCACACCTACAACCATCTGATCTTGAACAAAGCTGACAAAACAAGCAATGAGGAAAACACCCTATTCAATAAATGGTGCTGGAATAACTGGCTAGCTATATGCAGAAGATTGAAACTGGACTCCTTTCTTACACCATATACAAAAATCAACTCAAGATGGATTAAAGACTTAAATGTAAATCCCCAAACTATAAAAACTCTGGAAGACAACCTATGCAATACCATTCTGGACATAGAAATGGGCAAATATTTCATGATGAAGATGCCAAAAGCAATCAGAATGAAACCAAAATTTGACAAATGGGATCTAACTAAACTTAAGAGCTTCTGCACAGAAAACAAAAACAAACAAAAACCAAAAAACTATTAACAGAGTAAACAGACAACCTACAGAATGGGAGAAAATATTTGCAAACTATGCATCTGTTAAAGGTCTGATATTCAGCATCTATAAGGAAATTAAATTTACAATAAGAAAACAACTCCACTGAAAAGTGGGCAAAGGACATGAATAGACTTTTTTTTTAAAGAAGACTTACATGCAGCCAACGAGCATATGATAAAAAGCTCAACATCACTGACCATTAGACAAATGCAAATCAGAACCACAATAAGGTACTATTTCACACCAGTCAGAATGGCTATCATTAGAGTCAAAAAATAACAGACGCTGGCAATGTTGTGGAGAAAAGGGAACACATACACTGTTAGTAGAAGTGTAAATTAGTTCAACCATTATGGAAAGCAGTGTGGCAATTCCTCAAAGACCTAAAACAGAACTACTGTTGCTGGATTCATCCCAGCAATCGCATTACTGGGTAGGTACCCAGAGGAATATAAATCATTCTACTGTAAAGATACACACACATGCATGTTCATTGCAGCACTGTTCACTATAGCAAAGACATGGAAGCAACCTGAATGCCTATCAGTGACAAACTGGATAAAGAAAATGTGGTACACATACATGATGGAATACTATGCAGCCATAAAAAAGCAAGAGATTATGTCTTTTGCGGGAGCATGGATGGAGTTAGAGGCCATTAGCCTTAGCAAACTAAAGCAGGAAACAGAAAACCAAATACTGCATGTTCTCACTTGTAAGTGGGAGTTAAATGATGAGAACTCACGGACATAGAAAAGAACAACAGCTACTGGGACCTACTTGAGGATGAAGGGTAGGAGCAGCAAGAGGAGCAGAAAAAATAACTGTTGGGTACTAGGCTTAGTACCTAGGTGATGAAATAATCTGTACAACAAGCCCCTGTGACATGAGTTTACCTATATAACAAACCTGCACATGTACGCCAGAACCTAAAATAATAGGTAAAAATAAATAAATAGAAAGAAAAAAATTTGGAGATTGAAGAGGGGATGTTGGTGTGTATTTGTGCATACAAAGCCATGTGTAAGAGAGTGGCGGGAAGTGCAAAGGAGAGAAAACTGTTACAGATGCTTCTCATATCAGTGTGAGTGAGTGATAATGTCATTAGCTGAGATGGGGATGCTGGCGGAGAGGCTTAATCACCTGTATAAGTTGATGAGAGGGTGCACCCAGAGAGGAGGAGATTGAACAAAACAGGCTTATATTTTGAATGGTGACCAAGAAATGAGGGAGATTAAATATGGTAGGTCCATCCTCAGCAGACTCCTGGAAGTTAGAGGGCACTGGGACTTCACAGAATTTCCTCCAGGTCTGGTGAGAAAGAGGGCACAATGGATGCCCTCCTCCTCAGTCAACCTAAAGCAAAACCTACCACTCACAAATTCCCACTCTGCCCACCAGGCAAATCCCAATCAGCTTGTCAGGTCCTCTGATTAAAACACATGAACAGCTCAGGCTCACCAGGCATCTGAAGAAAATCTTTGGTAAAAAGGAGGCCAAGATAAAGCCACAGAAAATGTGGCCATGAACAAAACAAGAGAACCAGAAAGGGCTCTGAAAACTTTGAATAGTGTCCTTGGTGAAATTAAAAGAGATATTATATACATAAAACAAAAAGCAGCTTGTTGTGAAAAAAGAACAGCAAAAAAAGAAACTTATTAGAAATTAAACATTATTACACAAAAATCAACATTAGAACTAAAAGATAAAGTTGACGAAATAGCCCCGAAAGTAGGAAAAAGAGAACAGAAAATTTGAGAGTAAATGTCAATTCAAAAGTCCCAACTTTTGACTAAGTAGGAATTCTACAACTAGAGCACTGATTAAACAAAAGGGAGGAAATTAGTGAAGAAATGATGCAGAGAATTTACCTGGGACTAAAGATGCAAGTCCCTCAGTGCTCCAGAAAGTACCCAACACAGTGAATGAGAAAGGACCATGCCAAGGACATTATCATGGCATTTCAGAACTTTTTGGAAAAAGATTTTAAAACTAATGGGCAAAAGAAGGTCACCCAAAAAGGAACAAAAATCAAACTGGCATAAGAATTCTTAATAGCAACACAGGATAATAACTGAGCAGTGCTTTCATATTGCTAAGGAAAAACTTCCAACTCAGAATTCTATTTCTGGCCAAACTACCAATTGATTGTGAAAATGGAATAAATATATATTCATATATGCAAAGATTACACAATCTTCTTAAGAAGCTAATTGACAAGTGCTCCAGAAGAATGAGGGAGTAAAACATAAAAAAGCAACATGAAAAAGCCAAGAAAAAATGTCTACCATAGCAAGAAGTTAATCAACACGAACTACAATTGATAAATCAAATATCAGTGTAAGCACTATTTAGATATACGGAGGTAGAAGAGTTAACAGTTGTTGAATTTGGGGTGTATAAATGACAGTTAAAGAATTATACAATTGATTTCATTATAAGCTTTTTGGTGCATGTAATTTTTAAGCCATGTGCATGTATTACTTTGATTAAAATACTATTTATTAAAAAACTAAGAAATACATACCCTTAGTACATCTTATAGATACACTCAATAATTTGTGTTTTGGTAATATGTGAAAAGACCTATGTTCAAAGATATTCATTGCCCAAATGTTTGTAAGAGTGAAGGACTGTCAATTACTGAAACCTAAATATTCATCATTAGCAGAGCAGTTAAATAAATCATAGAACTATAGACATGAAAAGAAATAAGATATCTCCCTGTATGGTGATATGAAAAGAGCTCTGAACTATATTGTTCCATTAAAAAAAGCAAGGTGCAGGAGAGTGCAAAGCATGTTCCCATTTTTGTGTATTATAAAAGTTGTGTGTAGATATAGATGTGCTGTATAGTCATAGCAAACTTCTGGAAATTTACAAAAGTAACTGTTAACTATTGCCTCTAAGAAGTGGGATCAGGAAACTGGGAAGAGAGGAAGACTTAATATTCATTGTATGTGCTTTTATACTATTCATTGTTCACGTGTCACTGTACTCAATTTTTCAATGAAAAACTATCTTATATGAATTTATTAATCAGTTCATTAGCAAAAGAGTCTTGTCAGACAGTGCAGTGGGCCACTTTACAAATACTCCCTTCCTCTAGGGCTCCTGGTGTGATCCCACAGCCCTTGGAAAAACCAGAGTCGTTGGAAGGAAAAAGAAGCTTGGCTGTGGCGTACATAATTATTTCTCTGTTTGTTTCCAATATCTAGTGCCTGTTCCATACAATATGAATGTAAGCACACCAAAATTAATCCTGACTATAATTATACGAATTTTGACAACTTTGGCTGGTCTTTTCTTGCCATGTTCCGGCTGATGACCCAAGATTCCTGGGAGAAGCTTTATCAACAGGTTATCTATTTATAGTCTCTCTTTTTCCTCTCTCCCTCCCTCCATCCTTTTCCTTCCCCCTCTATCACTTACCTATTTATTTACCATTTATTTATAACTACCATTCATCCCCCACTTTTATCATCTGTCTCATCTTTAACAGTTGTACTGTTAAATTCTGCCTTACTAATTCTGTTAGAAACTTCAAGAAAATTGGCCAGGCACGCTGACTCCCGCCTGTAATCCTAGCACTTTGGAAGGCTGAGGCTTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCAACTCTACTAAAAATACAAAAATTAGCAGGGCATGGTGACAGGTGCCTGTAATCCCCACTACTTGGAAAGATGAGGCAGGAGAATCACTTGAACCTAGGAGGCGGAGGTTGCAATGAGCCGAGATCACACCACTGCACTCCAGCCTGGGTGATAGAGTGAAACTCCGTCTCAAAAAAAAAAAAAAAAAGAAACTTCAAGAAAATTTTAAATGCAATATTTGGAAGAAGACTCCAAAGAACCCCTCCTCTTCCTAGAAATGTTAGCCCCCTCCAGATTCCCTACTGCCCTATCTCTCTTGCCACTTTTATTTTAAAAATGCAGATACTCTTGAGAGAGTTATAGATAGTAAAGTCACTGAAACTTCCATGTGAAGGTGTGGATAGTATCTCCCCACTGAGAGATAGGGCATCAGGAATCCCTGGAGTATTAGGGAAGCTATATGGTCTGTGGTCAACTGAGCCCACCAGTGCTGGGGGAAGACCAGTTGTCTGCTACTGTGTGGTTTTTGCTTGAGGTGATGGTCACTTTCCTGTGGAAGAAGGTTTGATATTCAAGAACATGCTCTGTCCTTTGCAGACCCTGCGTACTACTGGGCTCTACTCAGTCTTCTTCTTCATTGTGGTCATTTTCCTGGGCTCCTTCTACCTGATTAACTTAACCCTGGCTGTTGTTACCATGGCATATGAGGAGCAGAACAAGAATGTAGCTGCAGAGATAGAGGCCAAGGAAAAGATGTTTCAGGAAGCCCAGCAGCTGTTAAAGGAGGAAAAGGAGGTAGGGACATGGGGTCAGACAGTGGGATCTGCTCTGGGGAGGGGAAGGGGTCCCCAGAGGCAATGTGACCTGAGACTCAAGGCTGCCACCCTTGGCCTGGTCTCAGTGCTTTCTCTACTGGTGCTTTTCTGAGAATATAAGTCCCTCGAAGAATGGCTGTGCTGCTATCTTTCACAAAATTGTCCAGGCACCAGGCCCTTCAAAACGGGGAACGCTTGGTCTTGATGCTTTTTGACCTTCTGAAGTCTAAGCCTTCTAAGACATGCCCTAAATTACTGACTAGACGATACAACATAAGTATATGTAAATCCAGGCTGTGCTCCAGGCTTTCTTTTGGAAATCTGGAGATGTTTGTGTTCGGCGCCTTTGCTTGAGGTGGACTCTAGAATAATGTTCACCTCTCCCTGTCTGCAATCCTTGTCTTATTTATAGGCCATTCTAATAGTTTCCTTGGAATCTTTACATTCTTCCAGCTCAAGTCTCAGATTTTCAGGACAGGGACCATGTTTTATACTCGCACTGATCCTCTATAAGATTTGTCAGAAGGATGCCCATCGTAAGTGCTTAACATCTGCACTATCAGGCATAAGTATTTATTGAGTGCCTAGCGCATACTGGACACTGTGCTAAGGGGCAGTGACACAGAGATATGAAAGAGTCAATGCTGCTTTTGGTGGCTGCAATTTCTGTGCAAAAACAGGACATGTTTCCCCAGATAAATAACAGAGCTAGCGTAGTCCAGGTAGATGATGAGCAATTTAAATGATACAGAGCTACCAGAAGAGAGATTTTCACCACTCTCAGGTCGGGGTCATTTGCAATGTTTCATGAGGAGGTGTAATCTGTTCTTTAATTGCTTTTGTTCAATTTGAAGGCTCTGGTTGCCATGGGAATTGACAGAAGTTCACTTACTTCCCTTGAAACATCATATTTTACCCCAAAAAAGAGAAAGCTCTTTGGTAATAAGAAAAGGAAGTCCTTCTTTTTGAGAGAGTCTGGGAAAGACCAGCCTCCTGGGTCAGATTCTGATGAAGATTGCCAAAAAAAGGTAAGTCTTTTCCAGGGAATTAATGATACCATATTGCTGTCCAGGGGGACAGGTCCAATTGCCAATCAAATTGAAATAATTCATTTATTCAGTTACTCATTTATTCATTCATCCATTTGCACATACTTTCATGCATCCATGTGTCTTTTCAATTATCCATTTATTTGTAAACATCAATTTAGAGTGTTTACAGTGTTTACTATCTTCTTGGCACTGGGATAGGTGCTAGGGATACATGTTTTATACTCCACAGAGTGTACGGCTAATGATGAAGATATACTGATAAGAAGGCAGTTATGAGTCAGTGTGATGACTGATGTGCTGATTGCTGTGGGAGAACAGAGGAGGAACTATGTGTCAGTGTGGGCTCCCTCTGAAGCAAACCCAAAGACATTACAATGCAAGTAGATTATTTGGGAAGTGATCCCAGGAAGCACTGGGAAAGGAGGGAAGAAGTGCTATATGGAATGGAAGGTAACCAATGAAGATGCATTATCAAGCCAGTTACCACTGAAGCCAGGTAGTTGGAGTTTAATCCTTCCAAAGTAAGGGCATCAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATATATAGATATATACACACACACACACACACACACACACACACACATATATGTTGGTATATATATACCAACTTTGGACCATCATAGATTAAAGATTGCTGAAGACAGAAGACAATAATTTCCCTCTAATTTCCTACTTGCTGTGCACTAAGGAAGACAGGATTTGAATCATTAGAGAAAGTCCTCAAGTAAGGACATATGGGTGGGAAAACCAACAGCATCCAGAATTCATGGGGCCTGGGGGAAAAGCAATGGAAAAGGGTCCCCAGAAGATGCAGTGTGTCAGCTGAAAGGATGAATTCACCAGTAAAAGGAAAAAGAGAGCATTACCTGCAGAGAGAATAGAATGTGCAAAAGCCTAGAAGCGAAGGCAAGCAGGAGAACATGACCCATTAGAAAACTTGCCAGTGTGTGTGTATGCGCGTGCACGCACGCTCATGCATGCACATGTGTGATGAGGCTGGGGGAGGGTGGGTGGCACAAGGCTATAGTGATCAGTTGGGGGATCACAAAAGACCTCGAAAACAAAGCTAGAGAGTTTACCCTTTTCAATGAGGGCTAAGGGTAAAGAGCATTGAAAGGTTTATATAAGAGAGGGATATGACTGGATTTATGAAATTCTGGATGCATTGAAGACAGGGGATTGGAGGAGGGCAAAACTGGAGGCGACCAGTTAAGAGGCTGTTGCAGTAACCAAGTAAGAGGTGCCTGTGGCTTAAACCAGGGTTAGGGAAGTGAGGATGAAGAGCAATTAACAGACGACCGTGATCTGGAGAAGATAAAATTTAGCAGTTGACTTGATGGACAGTGTGGGGATAAGACCTGGTTTCCGACATGATTAGCTAACTTGATGGAGGGTAATTTATGGAGACAGGAAAGGTACACATTTTGGGAGTGGAAAGATGGCTCATTCAGTTTTGAATGTGCCAATTTCCAGATGCTTATGGGACCTCCAAGCAGGGATGTTGATTAGGCAGCGTGATGTGCAGAAATGAAGCTGGTGGTGGGGGAGGGATGGGCAGCAGAAAAAGTTATTCTTATCATTAAGGAGGGGGATGTTCAGACATATTTTAATTGTCCAGAGAAAAGTCAGAAGAGAGGAAGATGCTAAAGATACAGGTAAAGATGGCAAGAGCCACTTTCCTGAGATAGAGGGAGATGTAGTAGGTAGAAGGTGGATATGTATGCCTGATAATGTGGTGGGGAAAGGGAATTGAGAGGCCTTTGCAGAAGTAGGTAGAAGGATCCCATCCACTGCTATGATCAGTTCTCTGCTGTAGTAATAGGGAGAGATCTCATAGAAAGACTATCTAGATTGGCTTATCACAAAAAAGATAGTTCCCTCTGGGGCTCTGGTGAGTCGGAACCCTTGGAAAATAAGCCACTAGGAAAACCCAAATTTTCTGGATAGCTGGAGGTTTGCACTGTTTAATTCCAGTAACTTTAGAATTGTAGGTAGATGTGAGAGAGTGAGCTAGCCCTTGGGCCCAGCTTGAGAGTGTGTTTGAGGTTTAGGATCTAGATTTTGTGATATTTAGTGGGACTGTTCTTTTTAGTTACCAACCGTTTTAAAAACCGTATAGGCGGTTGTTCACTTCACAACTGCAGCTAGCACTTCACAATCTGGCTCCAGTCCACCTTTCTAAATTTATACTCCACTTCTTACCTTTAAACATTGTTTACAAATTGGATTTTGTATTGCTTTCATACCCGGAACCATTGCTTACTAGTTTTTGTAAAATTTTGTTTGCTCTTTGTTGGCAGTGAATGAAACACAGAGAAAGTAAAATGTCTGTAGGTAGGACTCTAGAAATGTGCATGCATAGGAAATATGAATGATCAAACCAGATTTCTCTTATTTGTTAAACATGAAATAAGAAAACTGAATATTTGGCATGCTGTAAAGACGAGGTGAGTAGACCTATAGAAAATGTTGAGCACTTTCATTACATGTAGCATTGGAACAAAGTAGTTTGTTAAGTTTTGAGGAGAGGCAGCCCCTGTCTTTAGAAGGTGCCCTATGCCTTCTCTTACAGCCACAGCTCCTAGAGCAAACCAAACGACTGTCCCAGAATCTATCACTGGACCACTTTGATGAGCATGGAGATCCTCTCCAAAGGCAGAGAGCACTGAGTGCTGTCAGCATCCTCACCATCACCATGAAGGGTAAGTTCCACATCCCAATCCAAGGGAAAGTCTACTTCAGTGATGTCCTTCCATTCTTCTTCTTCCCAATCCCCTAGAAGCCCTCTGCAAAGGAGGCTGTTTTGGAACCAACCCACTGTACCATCCTGCAAAGAGTGACTTCCTTTGCCTCTTTGGAGCATGTAAACAAAGTGGCTCACAAGAAGCAGACCTTCCCTGTGGGCCCACTCTAAACTGGCACCTGGGTAGAGGTGGAAGCTGGCTAGACTTGGGATGGGACTTCAGACAAAATGCATAAACAGGGCCTCAAACCAGGCCTGGGAGTCACAAGAGATCCAAGTGACAACAATAGTCAGTCAAAGAACAGAAGACTTTGAAGTCTCCAGGAGGTGGCAGTTGTTATTGAAGTTAGAGTCAGAAACCCCACATGGGCAATAGAAGACCTATGTAGAAAGGCTGGAGCCATCTGATGGACCAGATTAATAGCAGGTAACAGGACCCCACCCCAGGGGTTGGTGTTCAGGAGCAGGATTTACTCCCTGCAAGGGGGGTCAAATAGGTCCTCTAGCACTTTTAAGCCTACTGCATCATCTCGTTTGCCCTGCATGTGTCCCTGCAGCGGTGCCAGGGAAATCAGGGATGGTCTGAACTCAACTAGAGGATAGAGTCCCTTTTACTGTTTCAGTGCTTATAGATGCCCCTTCCCTTGAGCCCTGGTTGCCCCTTTTGCCTCTGTTACAGCTGACCTTATGGTCTGTCTTGAGTGGACATAACTTGTGCCTGTATCTATCTCTCTCTTGGATTCACAGCCAGCCAATGTTGATTTCCACATCTATATATCTAACATCAAAGCTCAGCTCCTGGATGCTCAGTAAGAGTGTAAATATTTGATGGACTTGACTGTACCGCATGCTAGTTGTATGTGGAACATTGTCTCTTCTGCACTAAGATTGGTGGAAAAACCTTTCTGACTACACAAATCAAAACATTTCACAACTGATCATGTATCAGGAGGCCCTGGGGAAGAGGGAGGAAGGAATGCACTCAGGCAAAGGGCATCTGGATTGCTTCAGAGCTAACTCCTCAACCTCTCGCTCATTCTCCTCCAGAACAAGAAAAATCACAAGAGCCTTGTCTCCCTTGTGGAGAAAACCTGGCATCCAAGTACCTCGTGTGGAACTGTTGCCCCCAGTGGCTGTGCGTTAAGAAGGTCCTGAGAACTGTGATGACTGACCCGTTTACTGAGCTGGCCATCACCATCTGCATCATCATCAACACTGTCTTCTTGGCCATGGAGCATCACAAGATGGAGGCCAGTTTTGAGAAGATGTTGAATATAGGGAATTTGGTAACATTACACTTTTTAAAAATAAAAAACATATTTCATACTTTAAAGTATAACTTTTTCAAAGTCTAAACATTTTGTGAATGAGCTAACCTAACCTAGGTTTTTGGTCCCCTTCAGTGGAGTGAGGGAAAAGATGGCTGTTACGGTTTTACTTTTACCCGGTTCATTTCAGAAACTGTCCATGTGTCCTGAGTTGGCCCTCTGGTATGTTCAGGTATCTCTTTAGCCATTCAGTGGTAAGCCAGATACAAGCAGACATCCTAAAAGTTCAGCTCTCTGAGATAATCAAGAAAATGTTAACCATATCCAGAGAACCGTGGTAATGGCTGTTGTGGTCCCAGTGATATTCATGACATCATTTGTCACAAAATAATGTGACTACTATTACTAGGTGTTATTCTCCCTGGAAAGATGAATCAGGGCCTGTCAGCTGTGGAAGTTTCACTTTTTTGGAAGGGGTTGTTTGGGGGTTGGTCAGGCTGGGGTGGGGTGAATTTCCAGACTTGAGCCTGCTTCAATATGTTTCTCATTGGATAGATCTAGTCTCAGTTAAATTGGGAAAACTAAGGGGAGCAGACTTTTCAAGGACTGTTCACTTCAGTTTATGGTTCTTGTGGAAGGCATTTTTCTCAAGGAAAAATTAGTCCCTCGAATATCAGAAACAACAAATAATTGCCCACGGGATGATCTACCCTAATTAGCTGATGGATTAATGAGCTAAAAACCTTACCAGAATGTCTAGCCTTTTCTTCTTAAACTTTTGTTTTATCTTTCTAATTTAAAAAATAATAAATGCTGTTTTATTTTTTAAAAATAAGGAAAAATATAAATAAGGGAAATAGAAGAAATTCATGTCCCATGTACCTGCTGACAAAGATGATCATTTTTAGCATTGCAGAATATTTGCAAGCTTTATGTAAGCATGTATGTATGTGTGTATGTATTTTTATTTAGTGTTATTTTTTCTCCTTTTTTGGCATTTTTTTTTTTGTGATGTCTTTTTACATGTCTTTAAAAAATTTGTTTTACATTGTTAAGAACCCACTGCAGCCGAGCACAGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCCGAGGCAGGCAGATCATGAGGTCAGGAGATCAAGACCATCCTGGCTAACACGGTGAAACCCCATCTCTACTAAAAATACAAAAATTTAGCCGGGCATGGTGGCAGGCGCCTGTAGTCCCAACTACTCAGGAGGCTGAGGCTGGAGAATGGTGTGAACCAGGGAGGCGGATCTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTGGGTGACAAAGTGAGACTCCATCTCAAAAAAAAAAAAAGAACCCACTGCACTAGAGTCAGGTACCTGTCATCCTTAGTTCCACATCCCTCCACAAGGAGCAGTGATACAGTGGAACTGTAGTATAAAACCAGGGTGGTTCTCCTCACAGGCAGTCTGCACATAGCACATAAATTCCTTTCTCCATGCTGCCTTTGGGAATAAATGCTTTGTGGGTGTGAATGGGTTAGACTAGATTCCATGCTGCTTGACAAACCTAACACATAGTTGCTTGTAACAGATCAGGGTCTCCTGGCCATGTATTCAGCTATCCTCTGCTGAGGCATCTCAGAAATGAGCTGAAATTGTTTCAGTTGAAGGCAGCAAGCTGTGTCTATAGGGGCATGGTCAGCAGACTCCACCTTTTAGCACCTCCTGGTGGAATGGAGTTACTGAATAAGTTGTAGAGAATACTCAAAAACCCAGAGTCATCTCTGTGGACAGGGCTTCCCAATTGCAGTGGTGATCTGTTTTCATCTGCTGGTCCAAATTTAGAAAATGAGAAAAACCCCCATCTCAGATTTACCACTGACTAGACATCCTGAGTGTGGAGGTGAAGCTACATTTTGCAGTTGGAAACTGAGTGATTGACATTCTTCTTCACTTCCTCCAGAGCAGGAGGAGCCACAGGGCCACAGGGACATTGTTGTTTTCCAAGTACACAGGCAGCAGCTGCACCCTAGCTCCTAAAAATTGGCAGGAAATATGTAGAACCATACACATAGCTTGTGTTTCCATTCCATGGGCCTACTCCCTGGACATACAATACCAAAATGGCAGCCACACTATTTACTGCTCCCATGGTAGTAGTGGTGGAGAGAAGCCTTGGAGTCTTAAGTTCCAAGGAGCACATCAAAACTTCCCTCCTCTGATGCATGGGCCAGGATTAAGCCACTCTGTAGAGTTCTGCAGGACTGGACTAGAAAATTAACTCTCTAGGGTAGAGATTGCTCTAATCAGAGGGATACACTGAATGCCAAGAAGCCATAAAGAAAAGTTGGATTGAATTGACAAAACAATGGTCCCAAGGTATCTGGTGATGCTGTAGAGCTTGATGCCTAAAGCCTAAAGGAGATACAAGAAGGAGGAGTGAGTCAAAAGAGGTGGGGATGACTACACAATTATCTAGGACAGGCATCACTGTATGCTTTTAGATACTGGTGGTGACACTGAGCAGGAGTAGATGGCAGTATTTAAAGGGGGAATATGAAAATAAACTTGAATTTTCTTTGTAACTCTTGATTTATTTTTATTTTTCTACCCTGGATTTATCTTTGCTATCCTTTTGTCTTTAACTTTTGCCATTTTATTTCAGGCATTTTTAAAAATAAATAGTAAATCATTGAATCATTTATAAAAATAGATAATCCATTATTTTTAATAATCGAATTTAGTCGTTTATAGGTTCCCCCCAGAATTGTAGTAGACAGTTTTATAACAATCCTTGTAAAGACTTTAATCCTTTTAATATTACCTTATTATTTTTGTATTCTTGCTTTTTTCATTTCTATTTATTTGTGTTTGTATGTGATAAGACTGTGCTGTTTGCCGTGAAGAAATTATTTGCTTTATTTTTTGCAAAGATATGGAAGACAGATTTATTTTTTAATTGGCTACTACATTCAACATTTTTACATATTTCTTAAGCTTGTATTTATCTGATTATTGAAGACAAAGACAAAACTATTTTGCCCATGGAAAATACAAAATTCAGTACTTTTTGTAATTCCTGCCTCTCCTTTTTATTCTTTGATATTTTGTTCTGGAAATGTGAGCCCTGCATTCTGAATGAGTTTTAGGATTATATTCTGATTCACTAATTCTCCTTTCAACCATAACAAATTTAAGGCTTATCCCATATATTGAGTTGCTCAAAGACTTTTTTTTTTTTTTTTTGCCCCTGGAAGTGTTTTCTAATTCTATGATCTGTGCCCCTTTTTTGTTTTTATTTTTAATTTTTCTGATCAAGGTGTTGAAATTAGGTGGTCAGGTGAACTCCTCCTGGATTCTAGTTTATGGGGGATAATTATCCTTAGTTTTCAGGGCTATTGTTTACATTCCTTCTCTCCCTATTTTAATTGTTTTAATTGGTATTCTAAAGAGAATTCAGAGGAGAAATTGCCTGACACTCTGTCACCCTGCCATCTACCAGTAATACCATTTTGCTTTATCTTCAACAGAATGAGAAATAATATTCATTTTCCATTCTGGTGTCTGTGAGCTTGGTTACATTGCTCTGGGCACCATGGAGAGGGATGGAGAGAGGAGCAAAGTCATAGTGAGGTTCATGCTACAATAGTAAATATTTCTATTGCATGGACTTTAGGGTATAGTGTGTCAATCATGAGAATACTTTTCATTTCATTGTAACCTCTGTACTTCCCTTGGGCCATTCTTAAAAGTGTTATCTCCGCAGCTTCCTTCTCTCATTCTTCTTTGTCCCACTCTAGGTTTTCACTAGCATTTTTATAGCAGAAATGTGCCTAAAAATCATTGCGCTCGATCCCTACCACTACTTTCGCCGAGGCTGGAACATTTTTGACAGCATTGTTGCTCTTCTGAGTTTTGCAGATGTAATGAACTGTGTACTTCAAAAGAGAAGCTGGCCATTCTTGCGTTCCTTCAGAGTGGTAAGGCACTTTCTTATTTTACTGCATAAACGTAGCTGCCTAAGTGGAAAAAACGTTTTGAGTGAGTTCAGTGGTTATCTTGTACTAAACTTTAATTTATCTTACCTTAACCCCAGAACCAAAACTGCACTCCTCTTCCTGCACAGGTCTTTAATAGTCTTGCAAGTCCCTGGGGGGACTTCGCTTAACTGAACCCTTATACCCTTAGCCCATTCCTGCCAGAGATGACCCTTTAGGCAACTGGGTACAAAATTGTGTCTCCCAGTGTGAGGACTCTGTTCCTTGCTCTGTGCACAACCCTTCCGACACTCTTTTTTTCCCATGGAGCTGTGAGGCAATAAATAAATAAGGGAAAATATTCCTATCTTTCAGGTCATATTGGGCTCGCCAAGGGGGGCAAAGACTATAGAATGCCGGGGAGGAATGCAGGGGAGCTGATGGCAGGTGCTATCTATCTCACAAGTTTCCTTCTCACTGCTGCCAGTGCTACATTTCTGTTCTGTAGTCTTTGCCTGCTATCCAGTTTGGAAGCATTCTGGAAACCTTTACAAAGTACCTAATATAAACTGACATGAGGGAAGGCACAAAGGATGAGGAAATCATTGTCTTTGAGAAGGTTGCAGCACAGGAGTGATACAAGACTGGGCTACATGCACAGTACAGTGTGACAGAGCCCAGGAAAGGTATAGACAGAGGACTACAGAGAAATCCAATCTGAGAAGGGGAACTAGAAAACCTCCAAGGTGGGATTTTGTCCCCTTTCAAAGATATTTACCTTTTTTTTTCAGATAGGATTTTTAAGCCTATAGAGTAAGACACCTCATTCAGTTACCTAAATGTACATTTTATATTTTTTATACTTGGGATATAGTAAGAGGTTACACTGCTCAAAGTCAGGTGTTATTAAGCTTTCCTACTAATGTTTTATTAGCACTCAGGTAAGCAAAATTGCATTTGCTTTATCATTGTTTAGGCTTATTACAGCCAGGCTCTAATGCACGGAAATCTGTGGCATTGTCTCTGTATGAGAATGGAACCACAGGTTTTTCCTGGGATGCTCCTAGCTCCCCTTTGAGTAGACAATGACAGTCCTCTTCAAGCAGAAGCCTTACTGACCTGCAACATTTCCATCCCCAAATAGGGGATGTGCTTAACAAAATTGTACTTTGGAGTAAAACCGTCAGTTTATATCAAAAGTTAATATAAGAAACAACCTATAAAACAAGTTGCTATGTTTTCTCTAAGGTTGTTTCCTATAAAGTCCGTGAAGATCTAGGATAAAATTTCTCTGTGGAAAAAGTATACAAGTCTTGAATAGCTGGTTGCCTTTTCTCTCCTGACTACAAATGCCATATTACTCTAATCATTCTTATGAGGCTTAGATTCAGACTCGTGGCCTGAATTATATAATGCAGAAGTTAGCAAACTTTTTCTGTAAAGGGCCACATGGTAAATGTTTTCAGCCTTACAGGCTGTGTGGTCTGTGTGGCAACCGTTAAACTTTGCCATTGTAGCACAAAAGAAGCCATAGACTTTCAAAAACCAGTGAGCATGGCTATGTTCCAATAAACTTTATTTATGGATGCTGAAACTTGAACTTCATATGATTTTAATGCATCATGAAATATCCTTTTGATTATTGTTATTTTCTTTTTTGAGACAGAGTTTGGCTCTGTTGCCCAGGCTGGAGTACAGTGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGCTCAAGCCATCCTCCCTCGTCAGCCTCCCGAGTAGCTGGGACTACAGACGCACACCACCACGCCTGGCTAATTTTTGTATTTTTTGTAGAGATGGGGGTTTCACCATGTTGCCCAGGCTGGTCTTGAACTCTCCTGTGCTCAAGCGATCACCCACCTCAGTCTCCCAAAGTGCTGGGATTACAGATGAGAGCCACTGTGCCTGGCCTGATTATTTTTCAATCATTTAAATGTGGAAAAGTATTCTTGTCTCTGAGGTCATACAAAAATAGGCAATGGACTGTATTTGGCTCTCAGGCCAGAGTTCGCTGACGCTTGGTATATTACATATAATTCCAAAATATTCATTTTATATAATAATTATGTAGCTCAGTCACCATGCTGATCCCAAAGTCCTTAATGATTAAAAAACTAAAACAAAATATTTCCTTTTTTTTTTTTTTTTATGAGATGGAGTCTTGCTCTGTCTCCAGGCTGGAGTGCAATGGCGCAATCTCAGCTCACTGCAACCTCTGACTCACAGGTTCAAGCGATTCCCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCATGCGCCACCATGCCCAGCTAATTTTTTTGTATTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGATGGTCTCAATCTCCTGACCTTGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGTGCCCGGCCAATGTTTCCATTTTCAAATAAGAAAGTAGATACAGGTGAGCATTAGATTATGACAGCGATCTGATGGCTATGTTCAGGATAGATTTAACTTTGAAGAGAGTTTTGGATGTCATAAGTTTGGGTATATGTCATTTGCTTTATAGATGAGCAGATGGAGTTTGTCCTTAACTGGCTTTTCTTCCATTTTTGTTTGTTGTCTTTTTCTACAGCTCAGGGTCTTCAAGTTAGCCAAATCCTGGCCAACTTTGAACACACTAATTAAGATAATCGGCAACTCTGTCGGAGCCCTTGGAAGCCTGACTGTGGTCCTGGTCATTGTGATCTTTATTTTCTCAGTAGTTGGCATGCAGCTTTTTGGCCGTAGCTTCAATTCCCAAAAGAGTCCAAAACTCTGTAACCCGACAGGCCCGACAGTCTCATGTTTACGGCACTGGCACATGGGGGATTTCTGGCACTCCTTCCTAGTGGTATTCCGCATCCTCTGCGGGGAATGGATCGAAAATATGTGGGAATGTATGCAAGAAGCGAATGCATCATCATCATTGTGTGTTATTGTCTTCATATTGATCACGGTGATAGGAAAACTTGTGGTATGTGTCTTAGATTTTTTTCAAAAAAAAAAAAAAGATCCTACATGCCTCTCAAATGCATTACTTCATTTGCAAAACTTTACTAGTAAAAATTATTATTCTAAATATAATGCCAAATAAATATTTGAAATATTCATTCAAATTTTGTTTGAGAAAAGTATGATAAGCATGAAAAAACCTCTTTGACACATATTACTCATTTGACTGATGCCCCCAAATTTATTTTATTCCAGATGATTATTCTAAGACAATAGTATTCTCTTCCCCAGTGATTGGCTGAAATAGGAGTTAGTGAGTTAATTTGGTCAATAGGATGTAAAGAGATGTCCACTGGAGCAATTTGGGAACAATCTTTTGTTCTTAAGAAATATGTGTTTTTAAAAAATGATCGTTTTACTTCCTCTGGGCTTTTTTATGACTGTGACCCCTGGAAAAGCCAGATCATGAGCTTAGAGCTGTGGTGGAATTGCCCACAGGAATATGGATCTGACATCCAGGACTGCTGTGCAATCCTCTCCTGTTTGCACGAATTGAGAGATGGTGTACTTTGTGCAGTGTCTTTAGGTCCCTGTTCCCCTAACTTCAATGAGGTATTGGGGCCTAGCCAGAGGGGTTTGGACCAAACCTGCAAACATTATGCAACCATGAGAAGAGCCAGCCTAAAACAAAGCCAACACTGAGGCAGGTAGAGTGAAAAGAGAGAACAAACCTGTATTCATGTCATAATTGGCCTGCTGTTCTATTTTCGAACTTCTTGTTATGCTACAGATTTTTGTTGTATAAGCTGGCTTGAGTCTGTTACTTGCAGCTGGAAGCATAGATATTTATGAGGAGGCTAATTAAGTGCACTTCATTGCCTTACTGCACAAGATCTTCAGGATCAAGTTGGGGTTATAGTGCAATATAATTAACTCTCTACACACGATAGCTTTATCCTAAGTACCTAATAAATGGTGTGCCTAGACACTAAGTGATACATACAAAAGCTCAGAAGAACAGGGCATCACCAGTGGGAGTGGGAGGAGAGCACCGAGAAAGGATTCGTGGAAGATTACAGAACTTGAGAAGACCACTTATATGGTAAGGAGGACTTAGACAGATAGAGGAGAGCTTAAGCAAAGATCCAGAGGTGGGAATTCTCACAGTGCAGCTGAGGAACTGTGACTAGACCCATTTACACCCACCAGGCACTTCTCTCCAAAGGCTGTCTATTGTCGAGCTGAAGAGAGCCACAAACAAAGGAATTTGAGGAGGTAAAGATGATGAATTAGAGAGTGTGTAATTATGAGCAAAGAACCCAGGTAGGAAGCTATTGGGATTATGTGAATTTGAAGTGATATGTGGTTCAACGAAAGAGTTGCATATTTGGTCCAACTCGCTTGGCTTGGCCCCAGTACGTTACTAAGGTTAGGGCAACAGGACCTAAAGAAACTGTAAAGTACATCATCTCACAAATATTCAAATAAGAAAGGACTGTACATTGGGCTTGAATATCAGATTCATATCCTTGTGGGAAATTCCAGCAAAAATCAAAGCTCACTATCCAGCTGTTCCTCAAGAAATGTACTCTCAATTTTGCCATATGGTTATCCCATTCTGCTTTACCCTGGTAAGGGTCTACTCATCCATATTATTTATATGTAGGGAAAGTGGACTTGGCTATTTGCACCCTTTAATTTCAATTTTTATTCTGTCTTACTGTTTCTATTTCTTTCTCAATCTTTTATTTATTGGATAGATGAGCTTTCTTAGGAGACAGTGGGACATAAAACTCTAATTTGATTCTGTTTTTAGTCTTGGGGAAAAGTTGTCCTGGAAACCCACTATATCTTTTCCTTTGCTAAACTTTCCTTTCTTCTTGCTACCCACCCCATTCCCAGGTGCTCAACCTCTTCATTGCCTTACTGCTCAATTCCTTTAGCAATGAGGAAAGAAATGGAAACTTAGAAGGAGAGGCCAGGAAAACTAAAGTCCAGTTAGCACTGGATCGATTCCGCCGGGCTTTTTGTTTTGTGAGACACACTCTTGAGCATTTCTGTCACAAGTGGTGCAGGAAGCAAAACTTACCACAGCAAAAAGAGGTGGCAGGAGGCTGTGCTGCACAAAGCAAAGACATCATTCCCCTGGTCATGGAGATGAAAAGGGGCTCAGAGACCCAGGAGGAGCTTGGTATACTAACCTCTGTACCAAAGACCCTGGGCGTCAGGCATGATTGGACTTGGTTGGCACCACTTGCGGAGGAGGAAGATGACGTTGAATTTTCTGGTGAAGATAATGCACAGCGCATCACACAACCTGAGCCTGAACAACAGGTATGAAGGTTCACACATAGACTTAAAGGTCATACAAAGCTGGAGTTATCACAGGGCACTGGTAGCCTGCCCTTTTCTAGGCACTATGCAAGGATAATAAGGGTACAAAACGTGGCTGCCCTTTGATACCCATGTGCACATCTCAGGACAGGTTCTTTATTGTGGTCCTGCAGGTGTGGCACAGATAGGAGGTGTCAGAAGAGTTCAGAGGGGAGCAAAATCAGAGTGGGCTGGGATGACAGGGGGAGTTCCTTTGGTTGAACTCTACAAGAAATGTAGAATTTGGACTGGCTGAAAAGTGAGGAAAAGGGGATTGGGAAGTAAACTGAATAGCATGTTTTGTTTGTTGGTTGGTTTGTTTGTTTTAGAAAAAAAAGGCAGTGCTGGGAACAAACTCAGTTTCTGCAGAGACCAATCTTGTGGGTGAGGAGTGGTGGGAGATGAGGGCTGTCAGGGAGTGTGAGGGTAATGGATATTGTATTAGTCTATTCAGGCTGCTATAACAAAATATTATAGACTGGGTAACTTCTAAACAAGAGAAATTTATTTCTCGCAGCTCTGAAGGCTGGGAAGTTTCAGATCAAGGCAGTGACAGATTTGATGTCTGAGGAGGGCCTGTTTACTGACTCATATTTGATAGCTATAAATTTCTCTCTAACCAATGTTTAGCTATATCAAATACATTTTATTTTCATTTTTATTTTCTATATATTTAATTATATTTTATTTTATCTTATTTTCTAATTCTTCTTATGATTTCTTCTTTGACCCACTGCTTATTTAAAAATGTGTTGTTTAATATTTATAAATATCTCAAATTTCTATTTGTTATTAATTTCGAATTTCACTCCATTGTGATCAGAGAACATACTTTGTACAATTTCAGCCCTTTTAAATTTTTGAGGCTTGTTTTGTGGCATAGTGTATTGTCTATCCTGGAAATGTTCCATGTGTACCTGACAAGAATGTGTATTCTGCCATTTGGAATTAAGTGTTCTATAGATGTCTGCTAGGTTTAACTGGCTTATAGTTTTGTTCAAATCTTCCATTTCCCTGTTGATCTTCTATCTAGTTGTTATTCATTCATTTCTGAAAATAAGGTATTGAGATCTCCAATGTTTATTGTGAATTGTCTGTTTCTTCCAACAGTTATGTCAATTTTCATTTCATATATTTTGGGTCTCTTCTGTTAGGTGTGTATATATGTTTATAAATGCTATATCTTCCCAATATTTTGGTTCTTTTATCATTATAAAATGTCCCTCTTTATCTCTAGTAAGTTTTTTGTTTTTAAATCTATTTTGTCTGATGTTAATAGAGTCATCCCAGCTTCTTTTATGGTTGCTGTTTGCATAACATGTCTTTTTCCATCCTTTTATTTTCAATCTATTTATATCTTTGAACCCAAAATGTGTCCTCTGCAGACAGTGTATAATTGGATCTTGTTTTCTTATCTATTCTGGCAATCTCTGCCTTCTGATTGGATTGGTTAAACCATTCATATTTAATGCTGTTGTTAATTCAGTTTATTTACTTCTCATATTTTCCTGTTTGTCTTCTATATGTCTTCCATGTTTTTTCCTTTATTATTTCTCTACTGCTTCACTTGCATTAAGTAAATATTTTCTAGTATAACATTTTAATAACTTTAGTTTTTTCAAAATCTTTTTGAATTATTTCCTTATTTGGGTACTCTAAAATTTGACATATACATCTAACTCATCAGAGTCTATCTCAGATTTATATTAATTCCAGTGAGCTATAGAAATATTACTCATATATAGCTCTATTTCCTCTCTCCTGTTTTTGTATTGTTTTAAATTCTACATATTATATTCATATATGTTAAAACCCAAACAACTTTTTTTTATTATTGCTTAATATAACTAGATGTCTACTGAAGGAGCTGAGAAAATAAAAGAAAATAAGTGTATATTTATAAGATTTATTTTAATAAACTTCTTAGTTACTCTTGGCTGTCTTCATTTGTTCCTGTGACTGTGAGTTACCTGCAGGTGTGATTTTCTTACTCCAATGCAACTTTGCTCCCACCCATATCCTTTGTGCTCTTATTAGCAAATATATTTTATTTCTATATGTTACATACCCAGCAACACAATTTTAAACATATTGTTTTACACAATTGCTTTTGAAATCAGCTAAAAGAAGAAAGGAAAAGAAATATATATTTTTTATCATACTTGTGTGTTGTATTACCATTTAGGGTCACTTGTTTTTTGCCTGTAGGATGTCCTTTAGTATTCTTTATAAGGTAGGCGAACTACCAAAGAATTCTCTGTGTTTTTCTGGGAATGTGATTATTTCATCTTCATTTTTGAAAGATAGCCTCACTAGATATAAAAATCTTGGTTCACTGTTGTTTTTTCTTTCAGCACTTTGAATATATCATATCACTGCCTTTTGGTCTCCATTATTTCTAATGCAAGGTCAGTGTTAATTTTATCAATGTCACCTTGTACATGATGAACCACTTTTCTCTTGCTACTTTCAAGATTTTCTCTTTTCTTTGTCTTTCAGCACTTTTACTATGATATGTCTAGGTGTGGATCTCTTTGTGTTTTTTCTACTTGGGGTTTCATTGGGCTTCTTAGATGTGGAGTGCTAGGATTTGAATGTGTCCTCTAAAGTTTATATGTTGGAGAATTAATTCCCAAAGTAACAATGTTGAGAGGTAAGACCTTTAATAGGTGATTAGATTATGAGGACTCTGGTCTCATGAATGGATTAATGCTGTTATTGTGGGAGTAGTTTAGTTACTGTGGAAGTGGGTTCCTAATAAAAGAATGAGTTTGCTGCCTTCCCTCCTTTCTCTTTCTTGCCATGTGGTGATGCCTTCTGCCATGTTATGATGCAGCAGGAAGTCCTTCACAAGATACTGGCCTGTTGATCTTGGTCTTCCCAGCCTCCAGAACTGTAGGAAATACATATCTGTTCTTTATAAATTACTAATTTCAGGAATTCTATTATAGCAGCATAAATTGGACTAAGACGTGTAGATTAATGTTTTCGATCAATTTTGAGGAGTCTTCAGCCATTACTTATTTAAGTATTTTTTCTGTTCCTTTCTCTCTTTCCTTTCTGGTTGGTGTGCTTAATGGTGCACCACATTTCTCTGAGGCTCTTTTTATTTTTCTTTCTTTTGTTTTCTCTGTGCTCTTTAGATTGCACAGTCTCTGTTGATTCATCTTTAAATTCACTGGTTCTTTCTTCAAATGTACTGTTGGTCTCTCTAGTGACTTTTTCATTTCAGTTATGATAATTTTCAACTCCAAAGTTTCTATTTGGTTCTTTTCAAAATATAATTTCTATTGATATTTTGTATTTGATTGTATATGGTCATAGTATCTTTTATTTCCTTAAGCATTGTACCTTTTAATTCTTTAAATATATTTATAATGGCTTCTTTGCATTCTTTTTCTATTAGCTCTGACATCTTGACCCTTTCATGTGCAGTTTCTGTTACTGACTGTTTTCCCCTGTGTGCGTCACACTTTCTTGTTTTTTGCACATTTCATATTTTTGTTTTGTTTTGAATTGGACATTTTAAGTAACATATTGTGGAAACTCTATAACTGATTCACTTTTCTCCTCTCTGAAGCTTCTTTATTAAATGGTAATCTGGCTAGACTATTTTAGTGGAATCTGCTCCCTCTCCAACCTATCCCCCTCCATTCACACACAATGTGAAAGCTTTGGTGTCACTCCTAGGAGAGTACATCCTTGGATGTGCACACGGTCAGTGTAGGATGACTGTTGTTTCAGCAAGGCTATCTTTGTCTCCTTTTGTGATCTCTCTGTTAAGCTGTCTGCCTCTTTTGGTATTACACTTCGATTTTAGGCTCAACTAATTGCTGGATAATTGCTCTATTTTCCTTGACAATGCCATGGGGCATAAATTGCTCAACTGTCTGATTCAATTAAATTCAGGAAGTGATAGCTTTTAAGCGCAGTCTTTGAGATTTGTTTTGACCCCAGGGGGACTCTCCTTAGCCATCACTTTCCGTAGTTCTTTGTGGTGAATTTGCTGACCTATGTTTTAGTTTCTTGCCTTTAATGAAGGAGAGCTACCAATCTCTTCTTATTTGCTTATGATCAAAATATCCAATATCCATTGTTTTCAAGAACACTCTTAGACTTATACTTCCCCACACCACTTTGTTTCAAATAAAGTCAGTTCTATGGAGAGCTTCAGAGCTCTTTTTTCTTATGAATTACCTCCCACTCTGGGCAAACTCTTTTAAGCACAATTCCAGGCACTTGGGCAGGGGCAGTAGCCTCTCAACTTCTCAGTTTCCTTCTTCCTTAATCCTCCCCACCCCCAGAGCTTTTGCCCTACAAGTAAGCTGGAATGAGGATGATTCTAGTATTCTTGGGCTGCCGCTTCTGGGATAAAGTCTCTGCCCTATGAGTGGAGGGCTAGATGGAAAAAGGGAGCCAACTTTTTGGCCACACTCACCAGAAATTTAGAGGCCTTAACTCAGAGTTGGAAGAGATAAGAAATGCTGATGGCCTGCAGCTGCTGCTGCTTTTTTTTTTTTCCATAACTCATGCATTTTTATTGGTAGGCATTTCATGGTGGTGTAATCATTTATGCATTGGAAAATGGGATTGTTTTTGATGATACTAAACAGCATTTATTTCTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTTTTGAGATGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCATGATCTCCGCTCACTGCAAGCTCTGGCTCCCGGGTTCCTGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATGACAGGCGCCGGCCACCACACTCGGCTGATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCTCTCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGCCAGCATTTCTTTATTTCAAATGCATTGAATGACCACCATGTGACAGGCCTTGCACTAAAATAAAATAAGTATGCAATAAGTAATTTTTTTTTTGAGGCGGAGTTTTGCTCTTGCCACCCAGGCTGGAGTGCAACGGCACAATCTCATCTCACTGCAACCTCTGCTTGCCGAGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGTGTGTGCCACCATGCCTGGCTAATTTGGTATTTTTAGTAGAGATGGGGTTTCACCATATTGGCCAGCCTGGTCTTGAACTCTGGACCTCAGGTGATCCGCCCACCTCAGCCTCCCAAAGTGCAGGAATTACAGGCATAAGCCACCGTGCCTGGCCTGCAATAGGTAATTTAACATGTAGGCTGTATTTTTTTTAATGTAAGCATTTAAAAAGATTTTCCCCTTATGCAGATATCATTGATGTGCCTTTCCATTCAAAAGGCTCAATATCTTTTTTTTCAATTTCATTAAAAAAAATTTAATTTCAGTAGTTTTTGGGGAATAAGTGGTTTTTGGTTACATGGATAAGTTCTTTAGCAGTGATTTGTGAGATTTTGGTGCACCCATCACCACATCCCTTGATTGAGAGCTAGGGTAGAGGGAGCCCTATCTTCTTGGCCACCTTGGTCTAGAGTGGAGCCTCCATAAAACTTGGCTGGAAGGGATGGGGGGAGAGGTCAGATCATGGCTTAAGTGTCATCAATTTCTGCTGTTCTTACTGAATTTTAGTAGATTTTCTTGAACAAATGTTTCTTCATTTGCTGTGCACTCTCAGAATAATTTCTAGAGACTTTAAATTATTTTTAAAACAGTTTTCACCACTTATGCTTATTTCCCTAGGGAGAACGTCCGCAGAGATTATCATGCTATCATCCAGGAAGTGAAACTCTATGTAACTTCTTTATTAAATTCTATATGTATTAATTTAAAGACATTTTCAGATTATTATGTAACTTTCATCTTTTTCTCAAGTGAATTCACCTCCTAGTTGTTAATTTTAGGAAGTCTACTGATTTTATGTTTGTTTCCTCATATATTTTGGAATGTTAGCTTAAAAGCTCATCTTTAGTGGGAAGATCTGTCATTCTCTTTGCAGTCTAAGTTCTATGATTGTCTGTAATAAATCCTTCTGTGGCATCCATTTCAGATCCATGTCTTCTATCACCAGCTTTGGGTTGTTGTCTAATAATGATACAAGTCATATTGCAGATCCAGTCACTGAACCAGTGGGTACTTTTGCCCAGGTCCCATTTCTGAGGTTGTACTTCTTCCTGTCTCCCTACGTATGCAGCTTTATACAATTGTAATCATAAGCGTTTGCTGCGACATTTGTGTTTTTTGGCTTTCTCTCTTAAGTGCAAAAACTTTACCTCAGCCCACAGTTTCAAGCCTTAAATATAGCTCTGGTTCCCTTCCTCATCTAGGGTACTTTTAGACACCATTACCTGCTATCTTATTTAAATTCTTACTACTTTATATTTCTTTTCTGTGTCTAGTCCACAGACAGGTTACCCCTTTTTGTATGTGTATGTTTGTGAGGTATATTCGTGGCTATATTTGTATGTGTGTGTATTTTTACATATGCTATTACATGTGTGGAGAAATGTTTCTGAATCATGACCTTATAATGTCAGAGTTGCAAGAGTCTTCACATAGATGTTAGGATAAAACATTCACCCAAGATGTGACTGTAACCAGCAAAAGGATCCGTCCGGCTGCTAGCTGCTTATGAAAAGAATTCAAAATAATAACAAATTATGATAAGAAAGAGCAATATTCTCTTATTATCTGTGCCAGCAAGGGGAAGAGCAGAGACCCATTCTACTCTTCAATTTGTGGAAGGAATGCGGGGGTTTTTATTTTATTTTATTTTTTATTTTTTTATTTTATCATTATTATACTTTAAGTTTTAGGGTACATGTGCACAACGTGCAGGTTTGTTACATATGTATACATATGCCATGTTGGTGTGCTGCACGCATTAACTCGTCATTTAGCATTAGGTATATCTCCTAATGCTATCCCTCCCCCCTCCCCCCACCCCACAACAGTCCCGGAATGCGGGGGTTTTTAAAGAAAGGGTTTGAGATGCAGAATAGGCAAGAGGGGCTAGGAGGTGCCAGGTGGTGTGACTTGCTCCAGTGGCCCCTCTTGAATTATTGTCCCATGTGGTGAAGGGGCTGGTGCCATCATGGATCCTGCCAGGTTATAAATTAGTTGCAGTCAATCTTGTAGTCACTCTACAGCCTGAAGTAGGTTCTGTTCTTGAAGTAATCTTGTTGGGGAGAGAATTCTAGAGGTACCTGGTCCCTATCAGGATCTGACTCTCGAAGCTTCTAAGAAAACATATGACCACATAAAAGAACATGGCGTGTACTTAAAAAGCATTTAGGTAAATAAATGTGCATAAGGTATGGGAATATAGTATGGGAAAGGAAAGGCAGTGGAGGCTCACAGCACATTCCAAGGCTGTATTTCAAGATAAGAGGTAACACATATGCAGGTTGTCTCAAAGTTGTATCTTGAGACTTGGAGGGGAAAGAGGAAGAGGAAAAGAAGAAAAAAATGTTTAAAACACAGTTCGAGGCTCAGCTGCCAAGCTGCTTGGTTACATGACTGTACACATCCTTATTAGATATTCCTGTAGGTCATCAGAACAAATTTATTTTTCAGTTTTATGTAGTGATATGTTTTATTATTTTTCTTTATGTCTTTTAGTTTCCTGTTTTGCTAAGGAAACTCTCTGTACCCCACATGATAGCAAGTTATTTTTCAAATTTTTCTTTTAAAATGTTTACCCATTTATTTTTACATCAGAATGTCATAAGATTATATATTTTATTTTTACTATATTATGGCATTGTGTACCATTTATTAAATAATTTATTATCTAGCCATTGAATTGAAAGAGCACATTTAAAATATATTAAATCCCCATGTAAATTATATTAAATCTCCATGTAAGTTTTATTTCTGAATTACTTGCGGAGTTTTGTACCAGAACCATACTGTTTTGATTATGTATCTTTGTAACAAGTTCAGTATCTGTTTCCTCTAGTAGTTTTCCCAGAATTATATTATGTTGAGTTTTTCCAGGAGCAGTTTCTAAAGAGAAAAAAGAGTAATAATGTCAAAGGCTTATTCTCATGACAAAGAGAAAACCAAATAGCATTCAAACTTCTTTCATCTTAAGTGACATGTCCAGGAGGAATATTAATTCTATTCTGGGCTCTCTGTGTTAGGCCTATGAGCTCCATCAGGAGAACAAGAAGCCCACGAGCCAGAGAGTTCAAAGTGTGGAAATTGACATGTTCTCTGAAGATGAGCCTCATCTGACCATACAGGATCCCCGAAAGGTAATTTTCCTAGGATGTTGTCAGAGGAACTTGTGGCTCATCCACACACCTTCTCCAGGATAGTTATTATGGAAAAATGGCACAATTAATGCAGAGAAAATTGCAAGAATCGAGTTTTGAGCAAAAGTCTAGGCCAGGTTTCTCAACTTTACTAGTATGACATTTGAAGCAGGACAATTATTTGTCATGGTGAGCTTTTCTGTGAATTGTAAGATGTTTATCCATATCCTTGGCCTCTATGCATTTGATGCCAGTAGCATCCGCCAGTTAAGGTAATGAAAAATGTCTCTAGACATTGCCAAAATGTCTCCTGGAGGGCCAAAGAAATAAGAACCACTGGTCTAGGCCTATTATTCCAGGAGGTGAGACAGTGGAAAATTATAGTCTCAGTAAAGTAGCCTTCTTCTCATCACATAATTGGAGTTAGGCAGCCTATCAGGGTTATGATCTGTAGGGAGAAAACGTGGAGGTACTGTATCTTCATATGCATTGCTATTCCTCAGCTATAGAGATGAGCTAGAGAGTTCTCTTCTGAAGTCAGGCTGGTCATAGCACTTAGAAAAGGACCCAGCAGGAGGGGTGTTTCTTGACACCTCTCATCCTAGAAACCTTTGCCTAGTTCATTTCATCCTTAAAAAATCAGACAATGAGAAACTCCGTACTACTATGGTGAAAGAAGGTCTTAGTAAAAGGCACCCCCTTCGTTTTGTTCTGATGTGCAGAAGTCTGATGTTACCAGTATACTATCAGAATGTAGCACCATTGATCTTCAGGATGGCTTTGGATGGTTACCTGAGATGGTTCCCAAAAAGCAACCAGAGAGATGTTTGCCCAAAGGTAAGTGGCAGTATTTCTGCATCCAGTTCACAGAATCTTTGGATGGGGTTTCTGTTCTTCTTTGAAGCTCTGTGGTTTTAGTAGATATCATCTTTCTACTTCTATTGGAGACAAAAGCAATGAGAGGGCACCCGTAGATTCTTGAGGCGATTGTCGGATCTAGGCAGAGGGGATTTCAGGAAATTCTTGCTTTAATTGGGCTCCTCTCACATGGCCCAAAATGCCAGGTAAACACTCTGGAAAACTCTCTTAGAAAGCCCACTGGGCCAATATGTCTGCCACTTTCTATGGTTCCCAGGGCATTCAATGTCAGGGTGTATTTGAGGTGAAAGACCAAGAAAAGAGTTTAGACATTGTAGTCAGGATCTTTAGCTTAGAAGCCAGACTGCCTGAGCTCAAATTCTGGCCTTGCCACTTATGAGCTGTGTGACTTGTTCAATTTAGTTAACCTCTCTGTGCTTTATTTCTAAAATGAGGGTAATAATAGTATCCAAAGCAAAGAGTTGTTGTGACTATTAAATGAGTGAATATAAATCACTTAGAAGAGCATCTGGAACAAAAATAAGCAATCAATAAATGCAGGCTTTCCTAATTCCTTGAGTGGATATAGTGGCTTTTTCCTATAGGCCTTTACTATTTGTTATATTCATTGTAGTGAACTTATTTGCACTGGTTTCCCCCATGAGGTCCTTAAAAACAAAGGCAGTGACTTCATCAGCATTGTATCTCTAGTGTAAGAATAGAAGCCGGCCCAGAAATGGGTTCAACAAATGTTGATAAATAAATAAGTGAAACAAATTAATGAATGAATCTAAAAAAATTATCTATAAGTTTAGGATTAAATTGGGTCCAGAAGCAACACAAATAACAGGTTTCAAACTCACATTCCAGGTTGCTTAACAGCTCCAAAGCTGGACAGGAGTTGGATTAAGGATTTCCAGTAGATTGCAGTTCTCCTCATTTATGTTTGCACTGGCTCTTGAGGTGTGCATATGATGTCAACAGTGGAGATGAGAAAGAAAGGTGGTTTTATCAAAGAAGGGAGGAGTGGGATAAAAGCCAGGAAAAAAGTGTAGTCTTTTAGGAAGATGAACCTACTGATGTTTTTGGAAGAAGAAAAGAGGCAGAGTGAAGAGAGCCCATAGTGGTTTAAGAAGATGAGGAAAGGAGCAGAATGTTATAATCATCATGAATAAGGATCAGCAGAATTTGTACAAGGGGAGGAGGAGAAAAGGTGGAAAGGGCAATGTGGTCTATATTGAAGGCCTGTGTGTGTTCAGAACAAATGCTCAGCTCTTTGTAAACGTCTCGACATTTGATCAAATTCTTTCGTGTATGACCTGAGAGGTTCACAAAGCCTGTGGCCATCCTACCTCATTCCCTGGAGTCCTCCAGACATGATCTCTTTATGATTACCAAAAAAATTGCTGCATAACAAACAACTACAAAACCTCAGTGGCATAAAATAGTAAACATGTGCCTGCAGGATTCAGCTGATCTTGATTGGCTTCATTCATGCAAGTGTTATCTAGCCTGGGCTCAACAGGGCAACATGGCTCTACTCCACATGTCTATCATCCTCCTTCCAGACCCAATGGGGTAGCCTGAATTTGCCTTTTTTTATGGAGATGGCACAAGACAGCAAGTGGAAGCATGCAAGGCTTTTCAAGACCTGGGCTCAGAACTGGCACAGTGTGACTTCCATTCCACTCTATTGGCTAAAGCAAGTCACATGGCAGGGACCAGATTTAAGATGTTAGAAAAAGATGCTCTACTTCTTTAATGGGAGCAACTGCAGGTCGCATGGCAAGGGGCATGAACACAGAAAGGGGTGAAGAGTTGCATTTTAATGCAGTTTATTACAGACACTAATGATAGTGCTACCATTAATTCCTTTCAGGCTTTGGTTGCTGCTTTCCATGCTGTAGCGTGGACAAGAGAAAGCCTCCCTGGGTCATTTGGTGGAACCTGCGGAAAACCTGCTACCAAATAGTGAAACACAGCTGGTTTGAGAGCTTTATTATCTTTGTGATTCTGCTGAGCAGTGGGGCACTGGTAAATCATCATTGTCTTGTGGAGACATTGGGCATCAGGGCAGCTGGGGAAGCTGCACTCTGACTTGGCTTCTCGTGGGACTGTGAATATGGGAGACCAGACAGAAGTGGTCTCTGATGCTGCTGGTTCCCGCAGTGGGCAGGGTTTTGCACAGCTTCTGAAATCGTGCTCAACTGGGATATCACCAAGCAAAGATGCTTTATCTGTGTGGAAGACTAACTAAAACCAGCACTTTAAAGTGGCCACTAGAGTCTGGTCCTTGATCATCTACCTCTCATTTTTGGGGGATTTCTAGACATTTTCACTGTATCCTACATAGTTCTTCCTCTAGACAGCAGTGCTTCTACTCTCCCTGTTACCATAAGCCATAGTGCTGTTTTCTGAAAGGTAAAATATTATGGGAGGGAGTTACAGGGACCAGATTAGGATATAATAAGTTGCCTTGGTATTTTCTGTCATTCATGATCCCTTGAAAGAAAGGACAAGGAGATGTTGCTTTATATGCATGGTCAAAGTGTGGTATGAGTCCCAATGAGATTTAGAAATAACTCTGAACCCATCAGCTTCTATAGCCATAGAGCTATCCACTCCTTTATGCTCTCTCTCCACACCTTCTGGTCCTTCTTGTCTCCCTTTCTCCTCCCTGCTAGGGAGGGATAGTATCTTTTCTCTTATCACCTGCATATTTCGCCCTCTCCTTTTCTATTTTCTGCTTTTTTCTATCATGCATCTTCCCTTAAAATGATCATACAGTAATGTGGAGCTAAATAATAGGGAACCAAATTCTCTTACTGTTTGACATAACAAAGTTATGAGTTTGTTATGAGAGTTTGGTTCCATATTACTAAACCCAGGAGCCTTGGGTTCTCCCCTGCTAAGAGTGACAGAACATTTCACAGTGAATCACTAGCAAGTGGAAAAAGACAAGGAAATGTCTTGCTCCTACACCCCATAGCTTCTGGTTAGTGTTTTACACTTGTGTGATCTCTAGGTGAAAAGAGATGAATACATACCAGAGCCCACCGGGGACTGTGATCAACATACATGTCCATTTGTCTACTTACTACAGTGTAAATTATTCTTCAAATAGAAAAGCAGATTTCTCTAAAACCATCCCAGAGAATTCCCTTAATAAATTTTTATTGTTAATCTTGGGTCTAGGGTTACCTGTAGACTTTCTGGTGACTACATTTTCACTGGCTGTCTACTGTTACTCAAGTGTAGGGTTTCTTGTATTTATTCCACAAATGGTACTTATGATATAGGCAGTGGGAAAGCAGTGACAGTGCCAAAAGGCCACAGCAAAAATGCCCCATGGTGCCATAAAAAGAAAATGTATGATCAGAAATGTAGCAGAGGGCACACAACTCCAGTTCTTATCTGCCTTCGGAAAGGCAGGTGTAGGCTGGGCAGAGCAAAGCTGAACACACAATGCAGAGGGTGCCAGGGGAAGGACCCAGCCCATATTTCTCTGTACATATTTGTAGAGAATGAGAACAAGAGACATCAAGTTGTTTGCCACTATATGTGTCTATCAGCAGCAGCAGCAGCATCACCTGGAAGCTTGTTAGAAACGCAGAGTATTACATTGTAACAAGCAGTCCATTGTAACAAGTTCCCTAGGTGATTTGGGGTTCATTAGAGTTTGAGAAACTCTGGTCTAGGCAACTATGTAATAATAAGAGCTTCCTGTGTCAGAAAATGGCCTAGATATAATGTGGAAGCCTCTGAAGCTCAATTGCAGGTATAAGCGTGCTCATCCATTGAGCTAACTCAGATCTGCTGAACCAGCTTACTGGTAAGCTTCCCAAATAAGGGAGGAAATGAGCACCCAATACCATTCAGATTTGTTTATCCTCAAGAGGCAAACAAACGAAGTCCCTGACCTCCAGGAGCCTGAAGTCTAGTAGGAAAGACAGCTGGAGTGATTATAGACCAGAAAGTATAGATCAAGATAGATAGAACAATTAAGTGGTCTACTCAGGGAAGCAGTGGGTGACATGGGAGCATGTAGGATGGGAACCTAACCCAATCCTGCTGGAACGGGACAGTTTCCTATAGAAAATATCAGCTAAGCTGAAATCTTAAAGCTGAAGAGCTGAAAGAGCAGAGGATGAGATAGAGGGCCTTCTTCAGCCAGAAACCAATTTGGCAATTGGAAGTGAGACTGTTTTACTCTTTTGCATTCTTTTTTTCACCCCAGGACTTTGGAGCTGTGGGCTTACCCTAGTCTCCAACAGCAAGGATGATTCCCTTCCTGCCTTCAATGTTGACGTAAATTTCATGGGCAGCTTTAGTAACAGGCCGTCTTCCTTTCTTTCATTCTCAGTAACTCTGTAAAGGCCAAAAGGACAGCTTATACGCTAGTTATATAGACCCTGTAGAATCACATACAGTTTGCGTGAGGGAAGTGTTTGGATGTAGCATTAAAAAGGGTCTCTCTGAAAGCCAGGATCTCTTTTTCATAATTACAAATAACAATGTTTAATATTCAAAGTAATTTACACTGGGAGCTTTAGCCTAGAGTGTTCCAAATTTTGTTAATGTCATGGCACACATAAGAAATGACACTATTTGTAGGGAATGGTAAGGTAAATGGATAAGGCTGTTTGTAGGGATAGATGAGGGGCATCAGGGTTGGCAGGATTTAGAGAACGGGCTAAGGCACTTGGACTTAATGTTATAAGCAATAAAGAGACACTGAATTAATCCACCAAGGCAATTGGGAGGATGAGAGATGGTCTATGCCACTGGGTGGTCCTGGGAATGTTGGCAAGACATATTTGGCAGGAGTTGAAAAGATGAGGAAGCCCAGGAAATAATGTGCTGATCCATTAGGAAGATGTTTTAATATTCAGGGGACCAGTAATAAGCAGTTGAGGAACTATTGTGACAACAGAAGAAAGAAGATTTTCCTTCTTCCATTTTTACTTACTCAAGGAATGGATTCCATTGGAAAAGTTTAGCCTTAATATCTTTTGAGCAGAACCTCATTACGAAGAGAGATACCAGTTTTTATTCATATAAGGAAGAAAAACAAAAAGTAAAGTTACCAAGGAGTTCTTTGCAAATATTCAACCTGGCCTATGGCTATGCTTTTTCATTTCAGATATTTGAAGATGTTCACCTTGAGAACCAACCCAAAATCCAAGAATTACTAAATTGTACTGACATTATTTTTACACATATTTTTATCCTGGAGATGGTACTAAAATGGGTAGCCTTCGGATTTGGAAAGTATTTCACCAGTGCCTGGTGCTGCCTTGATTTCATCATTGTGATTGTAAGTTTACCATCTCTGTGTCCCACACAGGCTGGGGTAGTGGTTAAGGCAGGGATCATATGGAGGCTGCTTTTCCTGTGTGCCGCATATGGATGTCTGTCTAAAATAGTTTTTCTTGTTATCATTGCCTCTTCACAGTACTTGTAAGTAGTTGTCATTTGTCTGTAAGCAGTTTAGCCTTTCCTAGGTCCCGCTCATCATACACTACACTTTATATGCTCACCACTACAACCTTTCCCATGATGGGATTCATTCACTTCTCTCCATAAGCACTGTTTATGTATAATATGTACCTAGAATAACTTTCAGTATCCTCGATTTTTTCTTCTTCCAACTTTCTATTTCTTTTCAAGACTTCTCAAAGCTGGTCAGCAGCCAATAGACTAGGCTAATATCCATCTTAATTGTCAAGCAGCATAAGAGGATTGTTCACATACAGTCTCTCATTTAATATGCATGACAATGTTCTGAATTAAGCAGCATTGTATGACACCTCCCACACTGTATACAAGGAAACCTGAAGCTCAGAGGATTATCAATTTGTTTCAGGTCATAGAGCTGATAAGTGGTGAAATTGGGATTCACGTGTCTAGCTGTATGACACAAAAGCTAATAATAGTATTAGTGGATCACATTTATTGAGTGCTTTCTCTGTATCAAGTACTCTTCCATATGCTCCAAATGCCTTCTCTCTTTTAGTTGTCATAGCCACTCTGTGGCAGGTGCTACTATTTTTGTATATTCAGAGAAAGAAAGTAAGTCATAGAGAGGCTAAGAAACATGTTCAGGATTGCCCAGCTAGAAGTGGTGGAGGTGAGATACAAACCTCGTAAGCCTCACTCCAAAGCTCTTGATTTAACATTACATCCTTTTTTTAAGATTAATTTTATGTTTACTGATACATAGTAGATGGACATATTTTCAGGATACATGTGATTATGTAATACCTTTATATAATTAAAACTGGTGCAACCTAGTAAGTCTCACTCCAGGGCCCTTGATTTTAACATTACCTCTTTCCATCATGATGTAATTAGTTAAACTTTGTCCCTGCTATGCAAGGAAGTAGATAAGACAAATATCTGAGCATAAACCATGTGGTCAGGAAACCCCACTGGAGAACCATAATGTTGTTGGTTAGCACAAATAGACCATGTGTTCATTTAATATGGACATCAATATTCTGAGTTAATCAGTATTACATGACATCTACCACATTACTAACAAGAAAACCAGAGGCTCAGAGACAAGGAAACAGAAGCTCATTGTGGTTCAGGCATTTTAGAAAGGTATAAATGCCATAGAAACTACAACAGGATTCAAAGATGGGAATAAAAACATCCACGTTTGTCAGTCCTATTAATTTCTAGAGAGCAAATAACTCTGTACGTATTATCTTAATAGATTATAGTAAAAAGAAAACCCTAAAAACTGTGTGAACAGATGATTGTTATCTCCATTATAGGGAACTCTGTTTTACCGGATACAAGCCCTACATTTAGGAGCCTGGGTCCTAAAGCTAATTGGTCTACCCTCTTTTTGTGTAACCTTAAGCAAAACATTTCCCCTCTTTTGACAATAATTTTCTCACAGGTAAAAATGAGGATGTGGACTATAAATTGCCCAAATTCACTGAATACATAATAGAAAGCTAGTGATAGTTTCACTAGAAATCTAGTGATCGCCATTAAAAATATCAAAACAGTTGTCCAAAGCCACACTGTCAAAAAAGGGTACCAGGCTGAGATGGGTGGGTTTATTTATTTATTTATTTATTTATTATATTTCAATAGGCTTTCAGGGAACAGGTGGTGTTTGGTTACCTGAATAAGTTATTTAGCGGTGATTTCTGAGATTTTGGTGCCCCATCGCCCGATCAGTGTACACTGTACCTAATGTAGTCTTTTATCCCTCACACCCTCATTTCCCCTGAGTCCCCAGAGTCCATTGTATTATTCTTACAACTTTGTGTCCTCTTAGCTTAGGTCTCTCTTATGAGTAAGAACATAAAATGTTTGGTTTTCCATTCCTGAGTTACTTCACTTAGAATAATAGTCTCCAATTCCATCCAGGTTGCTGTGAAAGCCACTATTTTGGTCCTTTTTATGGCTGAGTAGTATTCCATGGTGTATGTATATAGTATATACCAAAGACTATATATATATGGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATAGTATATACACCATATATATACCATATATATACACCATATGTCTCTTTCTCTCTCTATATATATATCACATTTTCTTTATCCACTGGTTGATTGATGGGCATTTGGGCTGAATCCATATTTTTGCAATTGCGAATTGTGCTGCTATAAACGTGTGTGCAAGTGTCTTTTTCATATAATGATTTCCTTTCTTCTGAGTAGATACACATTAGTGGGATTGCTGGATCAAATGACAGATCTACTTTTAGTTCTTTAAGGAATCTCCACATTGTTTTCCATAGTGGTTGTACTAGTTTACATTCCCACCAGCAGTGTAGAAGTGTTCTCTTTTCACCACATCCACGCCAACATCTATCATTTTTTGATTAGTGCCATTGTTGCAGGAGTAAGGTGGTATCACATTGTGGTTATGATTTGTATTTCCCTGATAATTAGTGATGTTGAACATTTTTTCCTATGTTTGTTGGCCATTTGTAAATCTTCTTTTGAGAATTGTCCATTCATGTCCTTTGCCCACTTTTGGATAGAATTATTTGTTTTTTTACTTGCTGATTTGATTGAGTTCTTTGTAGATTCTGGATATTAGTCCTTAGTCAGATGTATAGATTGCAAAGATTTTCACCCACTCTGTGGATGTCTGTTTACTGTGCTGATTGTTTCTTTTGCTGTGCAGAAGCTTTTTAGTTTTAAGTTCCATCTATTCATCTTTGTTTTTGTTGCATTTGCTTTTGGGTTCTTGGTCATGAAGTCTTTGCCTAAGCCAATGTCTAGAAGAGTTTTTCCCATGTTATCTTCTAGAATTTGTATGGTTTCAGGTCTTAGACTTAAATCCCTGATCCCTCTTGAGTTGATTTTCATATAAGATGAGAGATGAGGATCCAGTTTCATTCTCTTACCTGTAGCTATCCAGTTATCCCAGCACCATTTGTTGAATAGGGTGCTCTTTTCCCAATTTATGTTTTTGTTTGCTTTGTCGAAGATCGGTTGGCTGTAGGTATTTGGCTTTATTTCTGGGTTCTTTATTCTGTTCAATTGGTCTGTGTGCCTATTTTTATACCAGTACCATGCTGTTTTGGTGCCTATGGCCTTATAGTACAGTTAGAGGTCTGAAAATGTGATGTCTCCAGATTTGTTCTTTTTGGTTAGTCTTGCTTTGGGTATGCAGGCTCTTTTTTGGTTCCATGTAAATTTTAAGATTGTTTTTTCTAGTTCTGTGAAGAATGATAGTGGTATTTTGATGGGAATTGCATTGAATTTGTAGATCGCTTTTGGTAGTAGGGTCATTTTCACAATGTTGATTCTACCCATCCATGAACATGAGATGTGTTTTCATTTGTTTGTGTCATTGATGATTTCTTTCAGCAGTGTTTTGTAGTTTTCTTTGTAGAAGTCTTTCACTTCCTTGGTTAGGTATATTCCTAAGTATTTTATTTTATTTTATTTTCAGCTAGTGTAAAAGGGGTTGAGTTCTTGACTTGTTTCTCAGCTTGGTCACTGTTGGTGTATCACAGTGCTACTGATTTGTGTACATTGATTTTGTATCCTGAAACTTTGCTGAATTCATTTGCCAGTTCTAGGAGTTTTTTGGATGACTCTAGGATTTTCTATGTATACAATCATATCATCAAGAGGCCCAGATGGTTTTGCAGGTAAGTTTTACCAAACTTCCAAGGAAGAATTAATCTCATCTTATTCAAATTGTTACAAAGAATAGACAAAAGAAGGAAAGCTTGCAAATTTATTTAATGAGATAAACATAATATTGATAATAAATGAATAGAACAGAAATGAATCAAATAAGAAAATAATCTTACAATTATATATCTTTGCAAATCACAGATAAAGCACTCAACAAATGTGGTTTACTATTACTATTATTAGCCTTGACATCATGCAGACTGTGGTGTGAATCTGAAATTCACCACTTATCAGCTGTATGACCTGAAACAAGTTGCTGATCCTTTGAGCTTCAGGTTTCCTCATCTATAATATGGTAGATGTTGCAAAGTGCTGCTTAGCTTGGGATATTGTTGTACAGATTAAATGAGAGAATGTATGTGAACAGTTCTATAATATTTATTGTCAATCAAGATTATATAAAATTACCATCATAATTTTTATTAGAAAAGCATAAACCACAAAAAGATACACAATTCAACTACATCAAAATGTAGTAAAACTTTTATTTTTGTATTCAACAATACTCTAATTACAGTTACAAACCAACTAGAACCTGGGAGACAGTATTTTCCACAACAGATCATTATTATTCACAACATATACAAATTTCCTACAGATTCACAGGAAAAAACAACCCAGAGTAAGAATGGGAGAAGTATACAAACAGCCAATTCACAAAAGGAATAAACGTATGATGAAATATACACTAGTTCTATCACCAGTTAGCACAGAAGTGCATATTAAAATGAGAGGCCTCTTTTTACTCATTAGATTGGAAAACAAAATATATTGTTTTGAAACATGGTCTCACTCTGTCATCCAGGCTTGAGTGCAGTGGCAACCTCTGCCTCCCAAGCTCAATTGATCCTTTTGCCTCAGACTCCTAAGTAGCTGGGACTACAGGCGCCCACCACCATGCCCAGCTAATTTTTGTATTTTTTATGGAGACAGGGTTTTACCATGTTGCCCAGGCTGGTCTCAGACTCCTGAGCTCAAGCTATCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGCTTGAGCGACTGTGCCCAGCCATGGCAAAAATTTTAAAGTTGGATAACATCGATATTTAGAAGGATATAGGAAAATGCCATGGCAAAAATTTTAAAGTTGGATAACATCGATATTTAGAAGGATATAGGAAAATGCGATCTCAAACCACTGCTAATAATTTAATTGGTACAGCCACTGTAGGGAATATTTCGCACTATATTAAGTAAAAATCACAAATGACCCAACTCACAATCCACAAATATCACTTGTTATAGATCCAAGGAGAACTCTTGCATATGTACCCAAGAAAAATGCACACATCTGCCCAGTGTACTGTTGTGCATAATACTGTCCAGTGTACTGTTTTGCATAATAGGAAAGAAATGGAAATAACCTAGGTATTCACCAAGGAGGAATGGATCCGTAAAACATGAAGATTTATATGCTGTACTATTCCTCAACAGTTACAAGGAGCAAACCATAAGAATATAATCAACATGATGATTTCAAAAACAGTGCAGAGTGATAAATGCAAGTTAGATTTTAAAAATAAGATAAAACTAGTAGAAGACTTCTCACTTGAAGAGTACAGTATGGTGGGAAAGAATGTGGGTGTTTCTATGTGTCTCACTGATACCTGCAAGTCTGATGGAACAGAAACTTGGGTAAGGTTTAAGGTAAAGAGGTGTATAAGGTCTCTGTAGGAACATCCCTTCTCACCAACTATGACAATGATTTGGTAAAACAGAAGAATTGCTAAACTTTGCCATCTGTTTTCATCTGGCTAGGAATAAGTGGAGTATTTGCGAGAAAGCTACTTAATAACTTAATAACTTAAATAACTCTCCAAAAGTGGTGCCATTGTAAACAAAAAGCCCACTAAAAACATTATAAATACACTGTAAATTGATTATAAATTTCAAAATCTTAATATTCTGTCATATTAACTCAATTTCTCTTCACTACTACAATGAGGTTGGCAAGATTTGTGAGGAATGTTTTTAGGTAATAGTGAACTCTGACATCAGCTTAGTCAGGACTATCCTGATTCACTTCCTATCCAGCTGAGCAACTCTTCTTCCAAGCTGAGCCTCTGAAAACTCTCCATAATAAGAATGGCTGAAGATCTGCAGCAAGCAGATTCAACTCAAGCTCAGCATTGATTCCATTGTGATAATAGAAGCAAATCCTAATTCCTCATATGAAAGCCAATACCTTCTCGAATTGTCCTCTACTGAGCATCTGTAAATATTGTTAAGATTTAATTTGGTATACTTAACAGAGCTTTCTAATACTCAGTTCTGTCCTATTGAGAAGAAGCATATTGGGAGAGAAAATAGCATTTAATTCCCCAATAAGGAGCTATTTGGATTAGGGGAAATATTAGCAAATAATTTATTTTTTCCATGTAACTTTTTATTTTGATATCATTTCAAACTAAGAAAAATGTTGCAGGAAATAGTATAATGAAATCCACATATTCTTTCTTCAGGTTCACCAATTATTGTCACCCATCTGCTTTGTCATTTTTTTCTCTCTTTTTTTCCCCCTAAACTGCTTGAGATTAAGTTGCAAGCATCAAACCTATTACCCCTAAACATGTCAATGTATATTTCCTATCAAGGACAATGTCTCACATGATTACAATACAGTGATCAAAGTTAGAAATTTAACATTGATTCAATTAAAATCCACATCCATTTTCAAATTTTTCAAATTCACCATAAAGATCTTTGTTATGGCAATTGACTATCATAATAAATTTCAATTGACTGCCATAACAAGATTCTTTATGGTTAATTCCCTTTATGCATCCACCTCCTACCCCCACTCCAATCCAGGATCCAATTCAGCACCCCAGATTACATGTGATTGTTATATCTCTTTGCTTTCCTTTAATTCACAACAATTTCTCGGATTTTCTATGTCTTTCACTAAACAAATAACGTGAGTGCATTGTTCCCACGTTTATGGACTGTAGCTTTGGTAGATAAGGAGAAAAATGGACTTTCCAGCTGGACACCAAGTCAGGTACATTATCTGTGGCCTCTCTTCCCATCAGGTCTCTGTGACCACCCTCATTAACTTAATGGAATTGAAGTCCTTCCGGACTCTACGAGCACTGAGGCCTCTTCGTGCGCTGTCCCAGTTTGAAGGAATGAAGGTACATTCTGCAGAAGAATGGGTAGAAGTTCAGTTAACAGAGAAAGGTGGAAAGACCAACAGTTCTTTTTGGGCTGAGATTTCCTTAAATTGCCAAGCTTTTCCTGGGTTACTTCCCAGCCTGCCCAGTGCTTAGAATTTGAGGGGTAGAGAAAAGCCTAAGATATACTTTCTACCCTAAAAGCTTCTGTGACAGCCAAGATGAGCTGTAGCTTTGCCTCAGAATCCTGGGGTAGGTGTCCATAAGTCTCTAACACTGTCATCACCAGGGCACTTCTCAAGGTATCCTCAGTGGCAACAGAGAGGACCCATTATGTAGGGAGCTGAGAGCAGGGCTTGTCATGGGGATGGGGCACATGTATGTGGAGATTAAGGACAGAGCTCCAAGAGATCAAGCCCTGCACATCAGGCCCATGCTGAGAAGTTGAAGAGCCTGGTGCATCCTACCCATCTGTTATGGTTTTCTTTGCTTTTGTTTCCATAAGGTGGTGGTCAATGCTCTCATAGGTGCCATACCTGCCATTCTGAATGTTTTGCTTGTCTGCCTCATTTTCTGGCTCGTATTTTGTATTCTGGGAGTATACTTCTTTTCTGGAAAATTTGGGAAATGCATTAATGGAACAGACTCAGTTATAAATTATACCATCATTACAAATAAAAGTCAATGTGAAAGTGGCAATTTCTCTTGGATCAACCAGAAAGTCAACTTTGACAATGTGGGAAATGCTTACCTCGCTCTGCTGCAAGTGGTAAGTACAGTCAGTATGTTTTTCACTCTGAGGAGAAAAACCTTCAGAGAATCATGAATTGTTTCTCAGCTAATAAAAATGCAGTGACTCCATTGTGATAATAGAAGCAAATCCTAATTCCTTATATGAAAGCCAATGCCTTCTGGAATTGTCCTCTACTGAGCATCTGCAACTTCATTGACTGCTGAGCCTCAGCTTCCTAGTGATGCCCCTTTAAGGATCTGTACATCTCACCTTGTCCTGTATGGCACCTTGCTGGCTCTCCACATCTCCAGGCAGAGAGGAGTGGAGCCTCAGGTGTAGGTCATGACACTTTGAACAGTTCCCTGTTTAAAGCACAGTTGCCAGGTCTTGAACAGGCAGTGCAAATACATATGGCATCAGAAAAGAACCCTTTAACTCTTAGCCAGTCTCTTTTCAGCTGTTGGGCCACAGCTTCAGCCTCTCTGGTAACTTTTAAGGTGGAAATTTCTTGTTTTTAAACTTTGTATCTTGTACAAATGAAGAAGAAGAAAAAAATCCAAGACACACACCCTTGAGTTCCCTTCTACTGTTCTTTTATTTGAGCATCCTGTCCTAGAAGGTTGGGGTATCATCTTATATATTCAGCCCACTGGGCCTCACCTGCTTTTCCATGCCATGTATCTGTTGATGGCAAGTCTACATTTGTATTATTCATGTTTTTCTGCTTTTGTTGCAGGCAACATTTAAGGGCTGGATGGATATTATATATGCAGCTGTTGATTCCACAGAGGTGAGTCAGTGTTCTACCATGTTCTGGAGTGTTATGGTCAAGTCAGAGATATCATGACTACATGGACAGTCCAGAACTGGCGTCATAGTTCCAGCAGCTGGGGTTCTCTGCCTTGTTTTCTTTGGAACAAAACACTATGAGATACCACTGCCTAATACAAAGGATTCTTATGCATCCTCTAGTGAGTATCTGATACCCCAGTATTCTCTGAAGTCCAGTCTTCTTTCTCCATGGTGGCCAAGGAAAAACCACTCATACAGGAGATGGAGCCAGAAGTGGCAGAATCACAGGATGCCTTGATTTTCTTCTTCCCTCTTCTCCAAATGGTTGGATAGTCAGAAACCAAATTTCAAAGAGTATCTGTTTTAGATTGTGGGAAACCTTAAGACTGCACTAAATTTGCAAGGGAGACCCTCGCATGGCTCCAGACTCCCTAGTGATGGCCAACTGTTGAATTTTAGAGTTTCTAGTGATGACCAGCTGTTGAATTTTAGTTAAAGAAAAGATAATCACAGATTCCCAAAGTTGGAATTGCACTCAGTAATCTAGCCTAGTTTCTATTTTCCAGCTTGGAAAGTTGAGGGATAAAAAGAAAGAGTTTCCCAAAGCCATAGAGTAAGCAGATGGTTGGCTCAAGAATGGAGTCAATGGCAGAAATTATTGTTTCCTATTATAGCACATGTGTGTGTTTCGACCTATTTGGGAGTCTGCAGAGTTGATGCCTAACACCTAAAAAGTATTCAGTACCTGTGAGCTGGTACAGACTGAGTATCCCTTATCTGAAATGCTTGGGACCAGAAGTGTTTCGGATTTTGGAGTTTGTAAGATTTGGGAATATTTGCATAATACTTACAGTTGAGCATCCGTGATCTGAAAATCCAAAACGCCACAGTGAGCATTTCCTTTAAGCATGACCTTTGCATGTTATGTCAGCATTCAAAAAGTTTCAGATTTTGGAGCACTTGAGACTTCGGATTTTCAGATTAGGGATACTCAGCCTGTAGTAGCAGTAGAAGTGGTTTGATTAGTGTTATAGCAGCACCGAGGGATAAGAATTAATCTTTATGGCCATTCCAGTTGTCATTATAAATTCACAGATTGTCATTATAAGTTAATATACAGATGATGCATAGCAACAAATCTGAGTTTGCTTAATTCTGGGTGTGTCAATAAAGTCAACTTTCTGGAAAGACAAAGGTAACAGTAGATACTTAACTGTTTATAACAATTATAGTTTACATTGTAATATTTATATATACAATGTAATTTTATAATTATATATACAACATAATTTTATAATTACAATTTATAGCAAAGGAAATATTTTAAAAATACTAATGTACTCATTTCTTTCGTCTTTTCGAGTACATGACCTTCACTCTAGTTTGGCTTTGACATCCATCTGTGCGGAGGTGGAGATGGGGAGTGGGGAGGAAAGGTTTTGCAGATGTGTAGTAGACAAGGTGTGTGGCTTGCAAATGCTCCAAGAGTGAGTGACAGGTGGGTGGAAGTGTGTGGAGGGGTGCCTGTTTTGATGCCGGTCCTGATATGTACTGGTTGTTGTATGCTGGCTTCTAGCTTGATGACTCTTGTCTATATAGTCACCCAGCACTCTGCTGGCCTCAAATATTGAAGTTGTGGTTGTTGAAACTGTGAGCGTTTTCCCCGTCATCTCCTCCTGGCCTTAAATACTGGTACTCTGCCATATCCTCCCCATGGTCCCTGGCAGTCTACTCCCATCCCTTGCTGCAAGGCCCAACATGACAGGCCCATGGCTATCAACTCCGTGTCCAAGCCACATGAGAACTGAGAGGTTCAAGAGACTTCTCTCTGTTCAATTATGTTACTCAAATCTCTTAAGTCAGCCTGCCTTCCTGATATATCTCAGGCCTTCCCAGTAGTAATTCCTTCCAGGAAATGCAGAAGTAAAGCAAGAATACCCTCTTTGCCTTCCATGTTTCCCTCAATTTACTGCATTCCAACCCCCCTTTCCAGACCCAAAGAGGGATAATCAGGACACACATCTTGCAGCTCTGATTTTCTGTCTCTGTTTATTTTTCCTACATTTCCTATGAACAAATTGGGTGAGCTTTGATAATTTCATCTTCTTGTCTAAATCTCCCTCCAATAAAACTCTCCACCTTCAAACCTTTGATTTAACAAAGAGGGACATGAAGGAGTACAGACATTCCTATATCTCAAAGCAGTAACCTAACATGCAAGACTATTAGCCCACTTGGCAATTTTATTTTAAATGTCTAATGCTAAATATTCTTTCCATTACGTTGTTTCTGTACTAATGTCTACCTTTTGTTCAAATAAATGGATGCCTCAAGCAAACTTGGACTAAAGTCAGGTAGTCTTCATAGCAAAAGAGAGAAATAATGTTTTCAAATAATTACCTTCATTACACTAGTCTACATTGTGAGTGTTGAATAACAAATACCATAAATGCATTAAACATTAGTCAGAGCTATCAGCACTTATATCTTTGATGTGATAGAGGGAAAAAAAGTGAAATCTTTTTGCCACCTGCCTGAAAGTGCTTGAAGCCAATAACAATGCAAAGGACTTGGATGAAAGGGCTCTTAGGTGATTTGGGTTGTATCTTAGACTAGGAGAGCTTGGTTGGAGTCCATTTGTGTCCATTTTAGAGCTCGATCATTTATTCATTCATTTAGATATTTATTCCATGTGTATCAAAAATCTACATATCAGAAGGTATGCTAGGCACTAGGAAGTTAAGAGTGAAAGGACATTACCACATATTAAGTAAAGCAGTGGGCTCTATATGGGGGTCATAGTAGTTCAACAACACTTAGTAGTTTAGTCACCTCTGCCCAAGGTGAACGTGCCCCTCCTCTGGTGGCCTCACCTCCAGTCAGGTTCCCTCCAGTTACATACCCATATGGACATTCCATATGGATGACTTTTTAAAAAGACAAATGTGATCATACCACTCATCTCCCTTACAGTCCCTCAATGATTCTCCTTTGCTTAGAATAAAAAGCCCGATTTTCCATCACCCCAGGTCCCCGCCACATGCCATCACTCAAGCTGAGCTGCAAAAACTGAAAGACAGGCTCCCAACAGGGGCTATGGCTGTTAGGAAGAGGCTATGTAGTCAATGTTGCTGCTAAGAAACACCTTGGTCTTCTAGATAAGGTAGTTAGAATGCTTATATTTTTCTCCAGTAATTGTTTTTTTCTCTTATTAAAAAAATTTCTAACAGAAAGAACAACAGCCAGAGTTTGAGAGCAATTCACTCGGTTACATTTACTTCGTAGTCTTTATCATCTTTGGCTCATTCTTCACTCTGAATCTCTTCATTGGCGTTATCATTGACAACTTCAACCAACAGCAGAAAAAGATAAGTATCTGGGTTGTCTTGATTTGGTAATTTTATCTCTGTCCTCCAAATGCATTAACAAAACATAATTCTGGCCTAGAATGTTATACAAAGTAGCCTTTATGATCTGCACAATAACAAGGAGGGAGCCTCCCATGTCTGCCTGGAGATGATCCTCAAAGATGAGGCTGAAAGAAGACTGAAAACATCTGGGTTTAATTAAGTCATTACTTTTATTCTCTTTTACCTTCTTAATCAATGGACATTGCTACTATTGAGATGAAACCCATTAATAACTGAACCCTAATAGAACAACAACCACCAGGATTAGTATTTATTAAATATGACACTAGGGTCATATTTTAATAACCTTTCAAATGCACTTTATTATCCACTATAAAACTAATATACTCTCCTTTTCCTGCCAGTGTGTCCTGGCCCCCAGCACCCAAGCCTCAGCAGATCTATCCACCCTACTCGGAAAGAAGATGATTGGGGAAGAGGCAGGGCTAGGGAGGTTTTCTTCCCTATGCATTGTAATAAACACATGTTTATTTTGCTGATTCTAAAATCCACAGGAGCTTTGTAGTGAATGTGGAAAATCAAGATAAAAAGAAAATTTAAATCACCCAAAGGTGGCCACCAAATGATAACCACTTTAGACCATGCCTCTTATGATTTTGATATAATCCATTCCAGTCACTTCGTATTACTTTTACACAGTTAGGATTACCTTATATTTTTAATTTTGTAGTTTTGACCTCATTAGTTATAAGCTTATGTCATAAAATTAACTTTGCTATAAGAATTCTCAGAGCCTCTGGCAGGGCGTGGTGGCTCATGCGTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGGAAATCACTTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCAGTCTCTACTAAAAATACAAAAAATTAACCAGCTGTGGTGGTACACGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCAGAGGTTGCAGTAAGCCGAGATCGCTCCACTGCATTCCAGCCTGGGCAACAGAGCGAGACTCCATCTCAAAGAAGGAAAAAGAGAAAAAAAAAAAAAAGGAATTCTCAGGGCTGAATTACATTGCATATTATGGATAGACCATAATTTATCCTGATCATTCCCCTCTTTGGACATTTATAGGTTGTTTTTAAATTTCTTGCTCTTCTAAAACAATTATGACCATTTTTAAAATTCTGTGTATCTCTGATTATTTCTTCAGCATAAATTCCTAGAATTACAATATTAAAAGCATTGAAGACATATTGTCAAAATACCTTCCAGAAATGCTGTGCTAGGTACCCTTATTCTGCAGAATATGAAAATGCATGGTTACTGAGATCTCATCATTCTAATTGTTATATATTTATTATCTTTTTTAGACTGGTGGGGAAAGTATTATCTCATTGTTTTATATTTATTTATTTGATCATTAGTGAGGTTACACACTTTTTCACAGGTTGATGGCCACAAGTATTTCTCCTTTAAGAGTTTTTTATTTATATCATTTGTCTATTTTTAAGTCTGAGATTGCATTTTTATATAAGTCCAATTTGTGACATGCATCTTATATTTTTGTAATAACATATTTAACTTATAGACATTAGTATTTTTCAGAGTCAAACTCCCAATCTTGTTATTTTTGGCTTTTTTTTAATTGTACTTATGAACTAAAGGCATTTCTCAATCCCCAAATCAAGTAACTAAATATTCTCCTTTGGTTTTCCTAATTGTTTTATGGCTTTCTTTTATTTTTCCTCTGGACTCTCATAATTCATCCAAAATGTACCTTGATACCAATTAGCCTTCTTAGCATCTGCTGGGAAAGTTGTTCCCTGTTGTTACACAAACTGTATTGCTTCTGCTACTCAGGAGTGTGTGATGTAGCATCCTCTCTGTAATATGCATCTTGATTGCTATCAATACCTGCAATTTGGATGTCAGTCTTTGTGATTTTAGATTTAAGCTTACATTAGAAATCATCGTTTTGAACAATCCACTATATCCTTTTGTTTTGTCTTGTTTTACTTTTTATTACAGAAAATCTCAAACATGCATAAGAGTGTACTAGAATTAACCTAAAATACCCATCACCTAGCACAACATTTATTGACTTAAGAATGTGCTTGTTCTAAGCCATATACGCCCTTACTCAACTCTGCCACCAGATTTTTTTTTATGGAGCAAACCACAGACATCACATCATTTGATTCATAAATATTTTAGAACACATCTCTTTTTAAACATAACCACAATTCCATTAGTACACCAAAAAGTTTAATGTGAGTTCTTTAAATTCATCAAATACCGAGTCTATTTTCAAATTGGTCTGAGCCTTTAATTTAAAAGAAAAATGTTTATTTATTAATTCTGTGTTTCATTTGCTTATTTGTTTCTGTTGAAGCTTTTTGTTTGTTTGTTTTCAAATCAGGATTCAAATACATAAGGTCTTTCCATTTGACAACTTTCTTGTTCTCTGAAATGCCACGATGTTCCAAGTTTAGGTTGTACCCTTCCTGCCCTAGTTCTGTAATCAGCTATTTCTCTAAAAACTGTTTGTTTCTTTGTGGGAAACGGTTTCCAAGACCACAGTCTAGAATGCTCATTGTCATTGTTTCTAAGCCTTTCTAGTGGACAGAGTTGCTTAAAATTCAGGACTACAAGGAAATATATAAGTGGAAATTTGCATACTGATTACTCAAATTCAGGACTACAAAGTTTTCACTTAACATCTATATCTCCCCCTTTCCACACTGAGAAACCTTATTCTCAAAGGCAAAGATGATAGAATTTAAAATTTCCACAATTACTCATTTTCTTTATCTCACAACACACATACACACACTACAGTCTCAGAATAGTGGTATTAATACTACTATGACATATATGATTACTAAAAGACTAATTTCTTCCCAGCATCCCAGAACAACTTGATTAATTTTTAGGTTGACTTCTAGGGCTACATATTTTTATCTGTGCTGGTGGAGTAAAGTCAAACCCCAAACCCTCATCAAAGTAAGCCTGAAGTCACAAATGATCAGGGAAGACTGGCTTTTTACTCATAGTCTGGATTGAGACAATTTTCTGGTTGTCTTCCATTACCAACAGACAGATTCGGGTTTTTTTCTTTACCCTTCTGTTGGCCACCTTTGGATAGTCCCATATTCTTGCAAAGACATCTGTGCCTCTCCTGTCTTTGCGTAGGCCTTATTTCCCATTACCCCCTTACATTTACTAAAAACTGAGATTCTAGTTTACAGAGATTAGTAAATGTTCTAAGACCAGCCATGGCTTTGGGCTAATTTACTAGTCTATCTTTTTGATCTCTCAATTTTCAGACCCCCAATTTTTTTCTTACTTTTATGCCAGTTCAGCTTTACATTTTAAAAGAATTCTGTTCTAGTTTATCTAGCCTTTCTAAATATTTTGCCCCCAAATTTCCAGATATGAGAATCCTAGTTCATTTTTCTATTCTTTCTTGCTCAGTAGTGAAAATAGTTAGAACTTTTTTTTTTCCCTGAGAGTGGCTCTGAACAGATTTTATGCAGCTTTGTTAGTAGTGTTATGTGTATTCATTATTTAAGAGAAATTCAATTGCCCTTTATGAGATGTTTGTCTAAATTTTTGTACTAATTTCTATTTTTGTTGCATCATGGTTTGATTTATGCAAGCTGAAATATATCAAATTTTTGTAAAGATTCTGTGTATATTTGCGAAGATTTGTAACCTTTCTGTTCTAAAAAGTGAAAAATAACTGATAAAGGTAACTTTATTTCTCCTGTATACCTTAACTTAAATCTTGTTTCTTTACAGCATTTCGCTATGGTCCAGTCTCTTAAGCTTACCATTGAAATTTTAAGAATTGTTAATAGAAATGTAATGTAACTTTAAAATTGTGTCAAAAAATTCAGAACTAGATTAAAAAAAAAACTGTTAAAGGTAATTAGCTCTTATCTTCTCCATATACTTAGGTGGCCAAGACATTTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAATTAGGATCCAAAAAACCTCAAAAACCCATTCCACGGCCTCTGGTGAGAGCAATTGAATGACTGTAAATATGTAGAACAGTTGAGTTACTGTGAGGAACTCAAATTTCCGTAATGCCATCCTTCTTTATTCTTAAAATTTGCAGACATTAAGGACTCAACAGAAGTCAAGACATATTCAAACACTTAGGAAAATTAGATACTCAAACTCAAAAGTAAGAGCCTGGACAACAACTTAAATTTATTTTTGAGTGAGCCAGGCAATATGTGCTTAAGTAAATACTTATTGCGACCATGTGAACTTAGTGGTTTGTTTGAACTTAATTTTGACCAGTTTATTTTATTTTATTTCAGTAGTTTTTGGGGAACAGGTGGTTTTTGGTTCCATGTGTAAGTTCTTTAGTGGTGATTTCTGAGAGTTTGGTGCACCTGTCACCCAAGCAGTGTACACTGTACCCAGTGTGTAGTCTTGTATTCCTCACCCTCTTCCCACCCATCTCCTTGATTCCCTGAAGTCCATTATATCATTCTTATGCCTTTATGTCCTCACAGCTTAGGTCCTACTTATAAGTGAGAACATACAATACTTGATTTTCCATTCCTGAGTTACTTCACTTAGAATAATGGTCTCCAACTCCATCCAGGTTGCTGCAAAGGCCATTATTTCATTCCTTTTTATGGCTGACTAGTATTCCATGGTGTAGATATACCACATTTTCTTTATCTGTTCTTTGGTTGGCAGGCATTTAGGTTGGTTTCACATTTTTGCCGTTGCGAATTGTGCTGCTACAAACATGTGGTATGTGTAAGTGTCTTTTTCATATAGTAATTTCTTTTCCTTTGGGTAGGTACCTAGTAGTGAGATTGCTGAATCCAATAGTAGTTCTACTTTTGGATCTTTAAGGAATTTCCATACTGGTTTTCATAATGGTTATACTAGTTTATATTCCCACCAACAGTGTAAAAGTGTTCCCTTTTCACCACATCCATGCCAACATCTATTATTTTTTTGATTTTTAAATTATGATAATTCTTGCAGGAGTAAGGTGGCATCCCATTGTGGTTTTAATTTGTATTTCCCTGATAATTAGTGATGTTGAGCATTTTTTTTCTATGTTTCTTGGCCATTTGTAAATCTTCTTTTGAGAATTGTCTGATTATGTCTTTTGCCTACTTTTGGATAGAATTGTTTTTGTCTTGATGATTTGTTTGAGTTCCTTGTAGATCCTGAAAATTAGTCTTTTGTCAGATGCATAGTTTGTGAATATTTTCTCCCACCTGGTGAGTTGTCTCTTTACTCTGCTGATTATTTATTTTGCCGTGCAGAAGATTTTTAGTTTAATTAGGTCTCATTTATTTATTTTTGTTTTTGTTGCATTTGCATTTGGGTTCTTGGTCATGAATTCTTTGCCTAAGCCAATATCTGGAAGAGTTTTCTTGATGTTATATTCTATAATTTGTATGGTTTCAGGTCTTAGAGTTAAGTCTGATCCATCTTGAGTTGATTTTTGTACCAGGTGAGAGATGAGGATCTGGCTTCATTCTTCTACATGCAAATTGCCAATTATCCCAACACAATTTGTGGGATAGGGTGTCCTTTCCCCAGTTTATGTTTGTTTGCTTTGCCAAAGATCAATTGGCTGTAACTATTTGGCTTTTTTGGGGGGTTCTCTATTCTGTTCCATTGGTCTACATGCCTGTTTTTATACCAGTACCATGCTGTTTCAGTAACTATGGCCTTGTAGTATAGTTTAAAGTCAGGTAATGTGATGCCTCCAAATTTGTTCTTTTTGCTTAGTCTTGCTTTGGCTATGCAGGCTCTTTTTTGGTTCCATATGAATTTTAAGATTTTTTTTTCTAGTCCTGTGAAGGATGACGGTGGTATTTTGTTGGGAATTGCACTGAGTCTGTAGATTGCTTTTGACAGTATGGTCATTTTCACAATGTTAATTCTACCTATCCATGAGCAGGGGATGTGTATCCATTTGTTTGTGTCATTAATGATTTTTTTCAGCAGTGTTTTGTAGTTTTCCCTGTAGAGATCTTTCATCTCCTTGGTTGGGTATATTTCTAAGTATTTTATTTTTTTCTGCAGCTGTTGTAAAATAAATTGAATTCTTGATTTGTTTCTCAGCTTGGTTGTTGTTGGTGTATAGCAGTGCTACTGTTTTATGTACATTGATTTTGTATCCTGAAACTTTACTGAATTTATTTATCAGATCTAGGAGCTTTTTGGATGAGTCTTTAGGGTTTTCTAAGTATACAGTCATAGCCTTGGCAAATAGCAATAGTTTGACTTCCTCTTTTCCAATTTGAATACTCTTTATTTCTTTCTCTTATCTGATTGCTCTGGCTAGGACTTCTGAGTGCTATGTTGAATAGAAGTGGTGAAAGTGGGCATCCTTATCTTGTCCCAGTTGTCATAGGGAATGCTTTCAACCATTACCTGTTCAGTATGATGTTGGCTGTGGGTTTGTCATAAATGGCTTTTACTACCTTGTGCTATGTTCCTTCTATGCCAGTTTTGCTGAGGGTTTTATCATAAAGAGATGCTGAATTTTGTCAAATGCTTTTTCTGTGTCTGTTGAGATGATCATATGATTTTTGTTTTTAATTCTGTTTATGTGATGTATCACATTTATTGACTTGCGTATGTTAAACCATCCCTGCATTCCTGGTATAAAACCTGCTTGATCATGGTGGATTTTCTTTTTGATATGCTGTTGGATTTAGTTAGCTAGTATTTTTTGAGTATTTTTGCACCTATAGTCATCAGGAATATTGGTCTGTAGTTTTCTTTTTTGTTGTGTCCTTTCCTGGTTTTGGTATTAGGGTGATACTGGCTTCATAGAATGATTTAGAGAGGATTCCCTCTTTCTCTATCTTTTAGTGTTAGTGTAACCACCCTTGCCCCCTGCCTAGTCAGAGCCAATTTATCAAGATGGGGGAATTGCAATGGAGAAAGAGTAATTCACACAGAGCTGGCTGTGTGGGAGATGGGAGTTTTATTATTACTCAAATCAGTCTCCCCGAGCATTCAGGGATCAGAGGTTTTTAAAGATAATTTGGTGGGTAGGGGCTAGGGAAATGGGGAGTGCTAATTGGTCAGGTTTGATATGGAATCATAGGGGGTCGAAGTGAGGTTTTCTTGCTATCTTCTGTTCCTGGGTGGAATGGCAGAACTGGTTGAGGCAGATTATGGGTCTGGGTGGTGTCAGCTGATCCATCCAGTGCAAGGTCTGCAAAATATCTCAAGCACTGATCTTAGGTTTTACAGTACTGTACTGATGTTATCCCCAGGAGCAATTTGAGGAGGTTCAGACTCTTGGAGCCCAGGGGCTGCATGACCCCTAAACCTTAATTTCTAATCTTGTAGCTAATTTGTTAGTCCTGCAAAGGCAGACTGGTCCCCAGGCAAGAGCAGGGGTCATTTTGGGAAAGATCTATTATCAATTTTGTTTCAAAGTCAAGCCATGAACTGAATTCCTTCCCAAAGTTAGTTCAGCCTATGCCCAGGAATGAACAAGGACAGCTTAAAGGTTAGAAGCAGATGGAGTCCATTAGGTGTGATTTCTTTCACTATCATAATTTCCTCAGTTATAATTTTGCAAATGCGGTTCCATAAGTAGGACTGGTACCAATTCTTCTTTAAATGTCTGATAGAATTCAGCTGTTAATCCATCTGGTCCTGGACTTTTTTTTGTTGGTAATTTTTTTATTACTGTTTCAATCTTGCTACTTTTTGTTGGTCTGTTCAGAGTTTCTATTTCTTCCTGATTTAATCTAGGATGGTTGTATATTTCCAGGAATTTATCCGTTTCCTCTGGATTTTCTAATTTGTGCATGTATGTGTTTGTAGTAGCCTTGAATGATCTTTCGTATTTCTGTGGTATCAGTTGTACTATCTCCTGTATCATTTTTAATTGAGCTTATTTTGATCTCTCTTCTTTTCTTGGTTAATCTCACTAATGGTCTATCATTTTTATCTTTCCAAATAACCAGCTTTTTGTTTCATTTACCATTTGTATTTTTTTTGTTTCAGTTTAATTTATTTCTGCTTGATCTTTGTTATTTATTTTCTTCTGCTGGGTTTGGATTTGCCTTTGGCTTGTTTTTCTTTCTCTAATTCCTTGAGATCTGACCTTAGATTGTCTGTGAGTGCTCTTTCAGACATTATGATGTACGCATTTAATGCTACAAACTTTCCTCTTAGCACCACTTTTGCTGTATCTCAGTGGTTTTCATAAGTTGTGTCACTGTTATCATTCAGTTCAAAGAATTTTTAAATTTCCATCTTGATTTCATTGTTAACCCAAAGATCATTCAAGAGGAGATTATTTAATTTCCATGTATTTGTATAGTTTTGAAGGTTCCTTTTGGAGTTAATTTCCAGTTTTTTTCCACTGTGGTCTGAGAGGATACTTGATATGATTTCGATTTTCTTAAATTTATTATGACTTGTTTTGTGATCTATCATATGGTCTATCTCCATGTGATATGGAGAATGCTCCATGTGCTGATGAATAGAATGTATATTCTGCAGTTGTTGGGTAGAATGTTCTGTAAATATCTGTTAATTTCATTTGTTCTAGGGTATAGTTTAAGTCCATTGTTTCTTTGTTTAATTTCTGTCTTGATTACCCGTCTAGTGCTGTCAGTGGAATACTGAAGTCTCCCAGTATTATTGTGTTGCCTCATTACTTAGGTCTAATAGTGATTGTTTTATGAATTTGGGAGCTCCAGTGTTAGGTACACATATATTTAGGACTGTGATGTTTTCCTGTTGGACTAATCATTTTATCATTATATAATGTCCCTCTTTGTCTTTTTTAACTTTTGTTGCTTTAAAGTCTGTTTTGTCTGATGTAAGAATAGCTACTCTTGCTCACTTTTCATTTCCATTTGCATGGAATGTCTTTTTCCACCCTTTACCTTAAGTTTATATGAGTCCTTATGTGTTAGGTGAGTCTCTTGAAGACAGTAGATGCTTGGTTTGTATCCATTCTACAATTCTATGTCTTTTAAATGGAGCGTTTAGGCCATTTACATTCTACGTTAGTATTGAGATGTGAAGTACTGTTCTATTCATCATGTTAGTTGTTGCCTAAATACTTTGTTGGGTTTTTTAAAATTATGTTGTTGTTTTATAGGCCCCGTGAGAATTATGCTTTAAGGAGGTTTTGTTTGTTTGTTTGTTTTGTTTTGTTTTGGTGTATTTCAAGGTTTGTTTCATAATTTAGAACTCCTTTTTGCATTTCTTGCAGTGTTGGTTTGGTGGTGATGAATTCTCTCAGCATTTATTTGTCTGAAAAAGACTTTATCTCTCCCTCTTTTTATGAAGCTTAGTTTTACTGGATACAAAATTCTTGGCTGACAATTATATTGTTTATGGAGGCTAAAGATAGGACCCCAATTCCTTCCAGCTGGTAAGGTTTCTGCTGAGAAGTTAACTGTTAATCTGATAGGTTTTCCTTTATAGGTTATCTAATTCTTTTGTCTCACAGCTCTTAAGATTCTTTCCTTCATCTTGACTTTAGATAACTTGATGACTATGTGCCTAGGTGATAATCTTTTTGTGATGAACTTCCCAGAAGTTCTTCGAGCTTCTTATTTGGTTGTCTAGATTTCTAACAAGGCCAGGAAAGTTTTCCTCAATTATTTCTTCAGATAAGTTTTATGAACTTTAGATTTCTTCATCAGGAAGACCAATTATTCTTAGGTTTAGCCATTTAACATAATCCCAAATTTCTTGGAGGCTTCATTCATTTTTTAAACTTTTTTCTTTGTCTTTGTCTGATTGGCTTTTAATTCAAAAGCTTTGTCTTTGAGCTCTAAAGTTCTTCTACTTGTCCTAGTCTATTGTTGAAACTTACCATGGCACTTTGCATTTCCGTAGGTGTTTCTTTCATTTCCAGAAGTTGTGAGTATTTTTTCTTTATGATATCTATTTCTCTGGAAAATTTTCTTATCCATATCTTGTATTGTTGTTTAAATTTCTTTAAGTGGGTTTTCACCTTTCTTTGGTATCTCCTTGATTAGCTTAATAATAAATCTTCTGAATTCTTTATCTGGAAATTCAGAGATTTCTTCTTGGTTTGGATCCATTGCTGAGAAGCTAGTATGATCTTTTGGAGGTGTTATAGAACCTATTTTGTCATATTACCAGAATTACTTTTCTGGTTCCTTTTCACTTGGGTAGACTATTACTTAAAATTGTTCTTGGATTTATTTTTAATTGAACTGTGTTTTTTAAATTTAAAATATTTTCCCTCTTTAACATCAGGACAGTGTGTATTTTAGCCTAATTTGATTATTGGTGCTTGTAGGAGTAAAGACTCTGTATGAGATCCTTAGTTATAAAGAGTCATTGTGCACTGGCTTTCCCTGATGCTGGTTGTAGTAGTTACTGTCTTAGTGTGCTGGCAAGTTCACTGTCTCCTATGGAGTTGGAATGGCAGGGATCTCTTGCAGCTTATCTGGTTCTCTCATGGTGTACATTTTTATTTATTTATTTATTTATTTTTTCCCAGTGTTTTATTTACTGACTTGATGATTCAGGCTTCAGGCCAATAGAGAAGGTATCCCTGAGTAGGCATTGGCTATGGCTAAGGCAGGTGGGTAAATGTAATACCCAACAGTGGGCTGAGGTCCCAGCCTTGATGAAGGTGGCTGGGAGACCTCTCAGTTAGATGCGCTGAGGTTTTATCAGGGTGAAGAGTAGAAGCTACCTCAGCTCCTCTGCCAGGTCAGCCAGAAAGCTATTCACCTCACAGCCTCACTCCTATCCCAGTGTTTTGGCTGTTCAGATCAGACAGCCACCTCTTTTCATCTGTAGGAATGTTGATGTTCCAAGTAGGGAGGAACTGTGACTCTGCCTCTCACACAGCCTGAATCTAGGGTGTGCTCCTTCTCTGGGGCCGCACTCACCCTGGATTGTTCCAGACAGGCTGTCTATAGATGCCTCCATACTGTGTTCCTGTGGGGGAAGCCCCAGCTGTGTCTGCAGTGAAGTGCCAGAGGAAAACAAGGACCTCTTTTCTAAGGCCCTTCACAATCACAGAGGATGCCTGCCCATTGGGCTAGAGGTGCAGACTTTCCCTACTGTGCCAGCACTGCAATTGTGTTTCTGCTGTGAGAAACTACCCACCAGCAGAAAGATCTGGAACTCAAGACCTGCTCTTCAGATTCTTTTGTCCCACAGGGTGATCCCCTGATGTGGTGCACTCCCCCTTCCCTAGGGATGGGGCTTCCTGAAAGCCAGACTGCAGTATTGTTATTGCTCTTCTGGTTCTAGCCACCTGGCAGGGCTACTAGGCTCTGGGGTGGTGCTGGGGAATGTTTGCAAAGAGTCCTGTGATGTGATCTGTTTTCAGGTTTCCCAGCTGTGGATACCAGCACCTGCTCTGGTGGAAGTGGCAGGAGAGTGAAGTAGACTCTGTGAGAGTCCTTGGCTGTAGATACATTTAGTATGCTGGCTTTCTCAAATGCTGGTTATGCTAGCAGTGAAGTTGTCACGTGGACAGACTCAGGACCTCTGGTTAGTCAGGATGTTGCAGGCAGTAGAATTAGCTGTCGTTTTCTCCTTCCTGGGATCAGGGCTATTCTGTCATGAATTGCAGTCATGTCCTGAGTTGGTTGACCTCCAGCCAGGAGGTGGTGCTTTCAAGAGAGCACCACTGTGCTATTAGCAGTGGGATATACGCTTGCCCTAAGTTGGCCAGGGGAGGTATTCTGACTTTCAGGAGACAGGCAGAGCCATAAAACTCCCAAGGGTTTATGTCTTTTGTGTTCAGCTACTAGGACAGGTAGAGAAAAACCATCAGGTGAGGGCAGGGTTAGGCAGGTCCAAGTGCAGACTCTCCTTGAGTGGGGCTTGCCATAACCACTGTGGGGGATGAGGAAGTGGTCCTCAGGCCAATGAGGTTATGTTCCAGAGGGGATTATGGCTACCTCTGCTGTGCAGTATAGATCACCAGAGAAGTGGGGGATAGCCAGTAGTGAAAGGCCTCGCCCAGCTCCCACACAGTTGGTGAGGCTGGTCTTGCTCCTGCAGTACCGTGCTAACTGCACCAAGTTTAGATCCAGGCAGCCTACTCAGGGATCTCAGACCTGCCCCAGGCCATAAGCTTCCCTGCTGACAAAGCAAGCATGGCTTTCAGGCCACATCCCTCCCTGTCTGCCCACAATGTCAGCAGCTCCTGCACTCGTATCTGCAGCAACTCCCGTTAGCCCCCTGGATTCTGCTCAAGGAAGTTTGTGCCCAGTCGAAATTATCACAGAATCTAGTTGGAAGTTTCTTTCACCCTATGGCCCCTCCTTTTATTTGGCTGGCTGCCTTCTCAGAAGACCCCTGTGAGATACAGTCAGGAATGGCATTCCAGGGCCCAAGCTGGAGACTGGGAGTACCTTCAAGGTTCCTGTTGCTTCTTCTACTTTTGCACTTCATGCAGCTCCCTAAAGTCATTTCAGCTCTAGGTAAGGTTACAACCTTCTCTCGTGATCTGCATTTTCAGATTCCCCAGTGGGGATGTGTGTTTGGAGGCAGGTTTTCCCCCCTCTCACTCACACTTTGGGAACTCACGGCTTTTCACCTGTCTCGTGCAGTCTGCAGTGGCATGTCACTTCTTTCAAAGGATCTGTGAATTCTTCTGGTTTTCCTGTTACGTTCCTGCGGTGGTTCTTGAAGCAAAAGTCCACAGTGTGAGTCTCCACACACTGTTCTGTCCATCCAAGTGCGAGATGCACGTTAGCCCTGCCTCCTATCTCCCATCTTCCTGTTTTTGCTTTTTTCAATTTTGATGAGTTTAAAATGTACTTGTAATACTTTTTACCAGTTTTTATTAGTGTGAGGTTAAGGTCATGCATACTTACTTCCACTTTACAAGTATTTATGATAAACTTTGTATATCAGGGATTGACAGACTTTTCCTTAAAGGCCCAGATAGTAAATATTTCAGTCTTTGCTGGCTATATGTCTCTGTCATAGTACTCAACTCTGCCATTGTGGTACAGAAGCAGCCATAGACAATATAGAACAAATAAACATGCCTGTATTCCAACACAACTTTATTTAGGGACATGGAAATTTGGATTTTATATAATTATCATATGTCGCAAAACTTTATTCTTCTTTTGATTTTTTTTTTCACCAAACATTTAAAAATGTAAAACCCATTCTGAGCTCATGGGCTGTACAATAACAGATAGGTCACTGGATTTGTCCTATAGGCCATGGTTTGCTGATCCCTGTGCTCTATGAATCCTGCTTTATCAGGTACTATGGGGCAAGATAGAAGTAGCATCCAGGGCAGCTCTGTGCTTGCAGTCTAATTGAGGAGCTGAGATGTTGTATACATACTTTAGAGCATTGGGTTATAAAGTAGTACAATAGGCACAACAGAGTTGGTAGCTCTGACAAGTGAGTTTGAATGCTCCATAGCAGAGCAGGTCAGTGGAGGCTGAAAAGTCAGGGAAAGCAGGATGAAAGAAGGGCTTGCAATCGGAAGCATGCAGATAATTTGGAGGTTTCCATTTGAACATGAGATCGCAAATGATGAATGCAGTCTACTTACTGCTATTCTCGGCACCCAGAATAGGGCCTGGCATAAAATGTGTTCTCAGTAATTATTTATTGAACAAAAAAATATGTTTTAAGGCACAAACAGAGTCCATCACACTAAAATGAAGTATTTTTGATGCACTAAAAAAGTCAATGAATGTCAGGGCCAAGGATTTAGAATTTATCTTTTATATAGTGAGAGATTTTTGACCACAGGGTTAATGATAAAATTATATTTGGGGAGGTTTAATCTGCCAGCGGTTTATGGGACTTCAGTTTACCTAAATTGAATCTAGCTTTTAAATACCAATCTCACTTTCTAGTACTAATTTCACTTTAAAATTCTTACTTGAACTTGAGCCAACAACAAGAAAAGAGAGAATTTTTGTTTACATATGCATGCTATACAACCACTGACTCATTATCAGTTGGTCTTGGGTTTGGCATTAACTAAAAGAGTCACAAGACACAATATGTTCTGACATATTTATCAGTTTATTTCAAGCAACACAGCACACAGCCCAACAAAAGTTGCAACACATAGATGCACTGCACAGAGCATGCTAGGCTGCAAGAGATGAACTGAAAGTGGGTGCAGACAACATTAACATAGGAAAGCTGTCACCTCCATTCCTTAGCCATCTTTTATAGCATGCTTATCACATGGCTTATCTGGAAGTAGGTGCAAAATCACAGCTCTCCCTGTGTAGCTACAACTCACCAATCAAGCCTTCAATGAATGATTCTGGTAATCAAATACAATCTCACCACCTAACACTGCACGTTTATCTTGACTTTTATATTCCAGATTAGTGTCGCATGAGATAAATCCAACATCATATCACCAGAAGTCCCTGAGTCTGGGCTTAGACTTTAAATCAACAGTTAGCCATTGACCCACAACTTTATCTCCCCAGAGATTTTTTTTCCATCTCTAAAATCCAAATATTAGCTTTTGACCCTCGAAACAAACGAAGAGCAATTGCATAGGTGGAAAACATAGCTGTTTTAGCCTGAGCATTGATGCATTTCATTTCCAGTAATAGAACAAGGGGTTTGGAATCACTTCGATTTCTCCTGAAGCATCTTCACAGGGTGGCTTCATTTCTTGGTTTAAAATTCATACTGAGGTATAGGAGTCACACTGCGTTCTGAAGTGACATAGAATGCTTTTTTTGATCATCCAACACTTTAGGTAATCCAACACTTTAGGTAATTTATGCTCTTAATCTCCTCTATTTGTCTAGATGTTAGAGTTAACTATAAAACCAATACTCACCCTCCCTCATATTTCTTTTTGCCCTTCATTCCGCTTCCCTGGAGTGCACCTTTTCTCCAGCTCCTTTCTGACTTCCCAAATTCATGCTTAAATAGGAAAGGCACTTGACTTTCTAGTGTCTTCTCCTGGCTCTTTGATAAAAGCCTTGGGGCTCCCAGCACATGGTTCGAAAGTCACAACTGAAATGTAGTGTCCTCCAGTGTTGTCCCTACCCTCCTTCCTCTCATTGCCCTTGCTCTCCCCAAGTGATATATAATTCACTCCCGTGGTTTTAATTCATATCAGTATTAATGTCATCTGTATTCTAGCTTCTGTACCTGCACTCCAAACTCATACTTCTACCTGCCCATTGCCCTTGGACCCCCCACTGGCACCTCAACTCAACCTGCTCAGGTCAGATCTTATCATCTCTCTTTAATATTCCTCATCCTCTGTTCTCTGGCTGGTTAGATGGTTTTATCAACCGGAAACTATATTCTCAGTCTTAACTTTCTCAAAGCCCACTACATTTGAATTCTCTGACGGTTCTTGCTTCCACCTCACATTAGCTCTTAAGCCTATATGCTTTTATCTTTATCTGCTGTCACCACCACAAGCCAACATCATCTTTGCCTGGATTATATAACAGATCCCTTGGGTGTCTAAAGATGTATTCTTAGGAGTATGTGAGTTCTCTAAAGATAATAGTGTATGCCACTTTGATTAGGTGATAATATAATATAAAACAATATGTACACAGATTGTAAAGCAACCAACGTATAGTCAAGAATGTTATAAAATTTCAATGTCTGAATTTGTATTTAGGAATAGTTTTCCACAAAGTGAAGAACAAAGTAGTGCTTCACAGCAGTTGTATTAAAGAAAATAATGGAATAATTGAGTAATTTCACTGCATATGGTAGACTTGGCATGCAAACTTGAGTTAGGACCTGTACCTGAACAATCAGCATATACTCACTTTTTGGGCAGTATGGCCATCTATATATAAACCACATGTAAATTCACCCTCAGTTATAGTATAAATTAATCATTTTTTTCTATTTTCAGTTTTAGTAATTATTGGATTATAATCTCATCTATTAGCTTTATCTTCAAGTGAACAAGAATTTTAAAATTGGCATTGAAGAATTATTTGAATACAGAATTTTGGTTAAGTTCTGATTATTGTGAATTGGCAAATGGAGCATTGAAGATACTAATTCCATTTTGTCCAACATACTTATGTGAGCAGGTATTCTCTGCACTAATATTGTTGAAGTAAGAATACAGAAACAGATAAAATATAGATACCAAGATTCATCTTTTTATTATTAAATCCAACATGCACTGTTTTATTTCAGCAAAGCACATCATCAATCACATCAAAGTTATGTTGTTACTAAGACTTTTTAATATTTTTTGTTTTAAAATGGAATTAAAGCTATAAGCCCAAGCACTTAAAACCAGTTACTAATTACATTTTAATGAAAATATATAAGGCAAGCCTGGGGATTTATAAGAATTTCTTGCCCTTTGAAAAGGGTCTGTGTATTTTTCAAGGAACTGCTTTAATTGTAAGGAAAAACAAATTCTGTTGTATTTATGTTCTTTTTATTGTATGTAATTTTATTCTTGCATTCAAAATGATTTATAATTTCTTTTTTTTCTCAGAACAAATGTCAAGGTCTCGTGTTCGACATAGTCACAAGCCAGATCTTTGACATCATCATCATAAGTCTCATTATCCTAAACATGATTAGCATGATGGCTGAATCATACAACCAACCCAAAGCCATGAAATCCATCCTTGACCATCTCAACTGGGTCTTTGTGGTCATCTTTACGTTAGAATGTCTCATCAAAATCTTTGCTTTGAGGCAATACTACTTCACCAATGGCTGGAATTTATTTGACTGTGTGGTCGTGCTTCTTTCCATTGTTAGTAAGTAAAATCAGCAGTCAGAGGGACTTTAAGAACCAGAAGTAAGTTTGTAAATCTTATCATTTTTTGAAGTTTGTTCAAACTATCCACAAAGCAGAAAACTGGGCCAAGTGTACTTTCTGAAAGAATAGACAGGGGTACTAATGCCATTCTCTACTGGGAAGTTGCTAGGAGATAGAGAGTAATTTCTGTTCCCTTAACTCACTACACAACTGAAATAGAGTTCAATAATCATGCAGCTAATGTATTCAATGGAAATAGACAAAATTAAAATGACTCAGAAGTTTTTGTGGTGGTAACCTGATTTATTCAGATAATTGAGTCAAAGTTCTACCTTTCCTGTGCAACTATAAGCCCTATGAGCATTTTTTAGTTTTAAGGATTTACTGCCCATGTTTTTTCCTTTGTTCAAAATCCACATGGGTGCCAGATCACAATTATTGTGCCCCTGTGGCACTGCTGTGCCCCCTTTCAACCCCTATACAAGGCCCCATGGCCCACTCTAGATTGGGCTTTCAAGAGTATATCATTCAGACAATCAAAGCTTTTTTTCTATTCTATATCCCATGTTAACTTAAACCACAGTGTGAACTGTGCTTGTCTCAGATTCTTTGACCCCGCTTACCAACCAGCCTCATTCTCTAATCAGTATATTTTTCCTAATCACACGCAGGGTTAACTAAAAGGCTTATAAAAGAATACAAAAGAAAGAAGGTTATTACCATACTCCTTCTTATAAAACACACACATACACACATCAACTTTAAAATTAAATTCTTCATGGCTAAACCATAAGTGCTTACTCTGGGTTTTTGAAGTCTGGTTTTAATTAAACTGTTAACTTGACATTTTATAATTATGTAAAACTGTCCAGGAGCCCTTCTCATAGTTCAAAACACAATGAAGACAAAGAAAACTGATTTTCCAAATGGAAGCTTTTTTGCGATCTCAGAAAGAATGCATCAAACAGATATATTGGATCAAGATATTTTGGTTCCTAATGCTACTGCAGCATTGCAGATGTTTCAGAGAACAACAACAAATATGCTACAGTTCATAGTGTGTTCTGGTTTCTAAGCCATGATGTTAATGTAGATAAAGCTTATCAATAGATTCTTCCATGAGTTTACAGTTAAGTAGACCTTTAATTTTTATTAGTCAATTCCAATCACAAGTCTTGGTGAAATTGACAGCAGCAAAAAAAAAAAAAAAAAAAAAAAAAAGGGCTAGAAAATGGTGGTCAGATGTCTGGGGAGGCCCTCTAAGAACTCAAAGAGCCCTTCCTCTGATGAGAAAAGAGAAGCTCATTCATTCCAACAGCTCCTTCCATGCAGTGCCAGGCATCATGCACTTTGCGGAAATGTTTCTGACTTTTGTCCTTTGAACAACTGCTTGGCAATTGCAGTTATACATGCTGTATGGCATTTGCCCCAGTGGATGAGAAAATCTACATATTAAGCCAAATAATACTCAGCGGTAGGATTAATCCATATATTAATCCTACTTACTAATGTGGACAGAGTTTTATATGCCTTTGCCAATATTTCTGCCGTCCAACGCTTTAGCCTCAGTACTTTTCCAAGCTCTTTCCTAAGTTTTTGGAGAAATGCAAAATGAGTGTGAGTTGAAATGAATCTCTCTCTAGGAGTTAACAATCTAGTTGAGGTATTACGGCTTACTCGTCTTAAAGTGCCAAAAAAAGATAGGGAAATTAATTGAACACCAAATCACATGGTGCAAAGCAGAAATGAGCTCAGAGAAAGAACAGATTACTATGGGCTTGATCAGCCCAAGAAATATTCATAGAGGAGGTAGATCTTAAATGATGGGCCTCCTCCAGTTGTTGCTGAATTGGCACTTAAGAAACTTTACATCTTTCCAGGGTTGACTTTCCAATTAAAGTATGAATTAAACTTCTTTGGCACAATTGGGTAGTCAGTGAAGTATATAATCAACTAGTTCTCTTTAGTCATTAGGTGACAGCAGAGCATTCTGCTGCCCATGAACCCTAATAGACCCCTAGTGACCTTACAGGACACTCAAGGTCATTAGGGGTCATGGGCACTCAAATACTGCCCATGACCCCTAACAACCCTAGAAGACTCTCAAAGCAAGAACAAGGCCATGTCTACACAAGACAAGCATGTTCCAACCATTCTATAATGTTTAGGCCAAAATGTGAATTGTCTTAAATCCTTGTATCACTTGCCTGCAGAGCAGGCAGGGCGACATTTTAGGTCAAATATTCAGCTGTTTGATCAAACCTCTGAAATGACATTTATGGGGTTTGCAGCAACAGAGAAAGTATCTCCAAATTGTGTCCCTTCAGCTTTTTGAGTTTGGATCTCAGAAGTCCTTGGAATGAGGATGACAATTATGAAAGATCCATGGGCAGCTTCAGCCTAAAGGTGAGCATTCCAATGCTACCATTTCCCTCTAGCCCTACAGACATTGTGGCTCTTTCAGCAGATAAGCTGTCCTCACTTGTAGTAGGGCTGTATTTAAATTCTTCTACACACAAATGTAGAAGAACTGTATGGCCCTTGGAAACTATTTTCCAAATTAAGACACAGATAGGTATTTTCCCTGATAGTGGTAAAAGAAATTCTTAGTTTTGTACATAAGCTACAAAGTATTGATAAAAAGTTGGCCCCCATAGGATTCTGAGGCAGGAGACAGGCTGGATCTGAAACCAGTGTGCCTTCTGGAAACTTATTTACTTTTCTCCTTCTCCTGAGGTACAATGATTTCTACCTTGGAAAATCAGGAGCACATTCCTTTCCCTCCGACGCTCTTCAGAATTGTCCGCTTGGCTCGGATTGGCCGAATCCTGAGGCTTGTCCGGGCTGCACGAGGAATCAGGACTCTCCTCTTTGCTCTGATGATGTCGCTTCCTTCTCTGTTCAACATTGGTCTTCTACTCTTTCTGATTATGTTTATCTATGCCATTCTGGGTATGAACTGGTTTTCCAAAGTGAATCCAGAGTCTGGAATCGATGACATATTCAACTTCAAGACTTTTGCCAGCAGCATGCTCTGTCTCTTCCAGATAAGCACATCAGCAGGTTGGGATTCCCTGCTCAGCCCCATGCTGCGATCAAAAGAATCATGTAACTCTTCCTCAGAAAACTGCCACCTCCCTGGCATAGCCACATCCTACTTTGTCAGTTACATTATCATCTCCTTTCTCATTGTTGTCAACATGTACATTGCTGTGATTTTAGAGAACTTCAATACAGCCACTGAAGAAAGTGAGGACCCTTTGGGTGAAGATGACTTTGACATATTTTATGAAGTGTGGGAAAAGTTTGACCCAGAAGCAACACAATTTATCAAATATTCTGCCCTTTCTGACTTTGCTGATGCCTTGCCTGAGCCTTTGCGTGTCGCAAAGCCAAATAAATATCAATTTCTAGTAATGGACTTGCCCATGGTGAGTGAAGATCGCCTCCACTGCATGGATATTCTTTTCGCCTTCACCGCTAGGGTACTCGGTGGCTCTGATGGCCTAGATAGTATGAAAGCAATGATGGAAGAGAAGTTCATGGAAGCCAATCCTCTCAAGAAGTTGTATGAACCCATAGTCACCACCACCAAGAGAAAGGAAGAGGAAAGAGGTGCTGCTATTATTCAAAAGGCCTTTCGAAAGTACATGATGAAGGTGACCAAGGGTGACCAAGGTGACCAAAATGACTTGGAAAACGGGCCTCATTCACCACTCCAGACTCTTTGCAATGGAGACTTGTCTAGCTTTGGGGTGGCCAAGGGCAAGGTCCACTGTGACTGAGCCCTCACCTCCACGCCTACCTCATAGCTTCACAGCCTTGCCTTCAGCCTCTGAGCTCCAGGGGTCAGCAGCTTAGTGTATCAACAGGGAGTGGATTCACCAAATTAGCCATTCCATTTTCTTTTCTGGCTAAAATAAATGATATTTCAATTTCATTTTAAATAATACTTACAGAGATATAAGATAAGGCTACTTGACAACCAGTGGTACTATTATAATAAGGAAGAAGACACCAGGAAGGACTGTAAAAGGACATACCAATTTTAGGATTGAAATAGTTCAGGCCGGGCGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGAGAGGCCAAGGCAGGTGGATCACGAGGTCAAGAGATCGAGACCATCCTGGCCAACATGATGAAACTCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCATGGTGGCGTGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTAAACCTGGGAGACGGAGGTTGCAGTGAGCCAAGATCGTGCCACTGCACTCCAGCCTGGTGACAGAGTGAGACTCTGTTTCAAAAAAGAAAAGAAAAGAAACATGGTTCAAATTATATCTAAACAAAAAAGAATAAGAAACAAAAAACACATTAAAATTTTAAGTTGTATTTTCTATGTTTCTAGATACATCATTTTTGTTTGATATTTTCCTGATGCAAGTATGTGGTTTATCACATGTAGCTCTTTTGCATGCTAAATGAAAATTCAAAACTTGCCAATAAATGAATAGCTTATTGCAGACATTTTTTACCAACATTAATTATTTTGGGTTTGTTTAAAACCTAGAGGCACAATCTTGACTTGTCAATTACTACCCTTTCACAAGCTACCATCTCAGATATATATATATATATAAATTCAATAAAGCTTTCTGTTTGTGTTCCTTTCAGGTAGTACACTAATATTCCCCTTTTGTTCAGTGGTTGTGTGAGTAGTGGTTTAAATGTGGTGCTTTGCTTGCAATTAAAGACTAGAACCAGTGGTGTATCTGACAAGTGTTTAACAACTGACTGTTCAGGAAAAAGAAAGCCATGCTTTATAGCATTCACTAATCTCTGTGGTGTGAGTACTCCCATTATGGCTGATTTCAAGCTATCAACATGTTACTGGACGTGGAGTTGAGAAGAGATGCTCATAATAAGTTCTTGAGAGCTGGCTCAAGCTGACTGCAGCACATCACTTGCTGAAACTGAAAAGAGAGCCCCACTTGGCTGGTTAAGATATTTGCAGTGGGCACCTTGGACAGAGAGAGTACCCTCTTAGCAACACGTGGGGCACGGGGTGAGAGATCCATAGACATACTGCTTAACCAGTGATCTTAGCCATTTGATGAGCCCTATTTACAGTTTTGGCTGAATGTCTGTTTCAAAGAAACACCCAGACCTATCAAAAGGGCTTGGGAGCAGGAGGAGCCCAGGAGTTCCCTAAAAACTGGAGATGCCAAGAGGTCAAACAGGCATTAGGCTTTCAGTCCCCCAAAAGGCTGGGCGAGGGGCAAAGAACCTACATTGTGATCATTGAGGAGTGGAGGAAAACCTCATAGAGATAGCCCTGGAACCCTGTATCTCAGACAGGTGAGAAGAACTGATGTGTCTGTCAGCAGCCTGGAGTCTGAGGTCGGGCCCTCTGACCTGCCCCACACTGCCCAGCAAAGGGAATGGCAGCACTGGATCCTGTGGTGGGCTGAGAGGTGATTATTTTCAGTGTGTAAGATCTTACCATGACTTGATGTATTCATTTCAAGGGCCTGAGTTTTGTAGGCAGCCTTGTGCTCTGAAGCATTGGTAAAAATAGTGATAATATCATTATCTAATATTTATTTAGTGGTTTCTGTGCATCAGGCACTGGCTAAGCATTTGTCATGAATGATCTCTTTTGTACCTCGTACCAACCCAATGAGGAAGATATACCCCATTTAAAAAGGAGAAAACTGAGGCTCAGAGAAGTTGAGTGACCTGCATACAGTCATGCACCTGGTGGAGCCCGAGCCTGTGTGCCATGATGGGAAGGGGAAGTGGGTGGGCTCAGGACCAGGGAAGCTTTGAGATGGGCTACCAGGGCTCCTGTGCAGGACTGGGAGGGCC

Example 5 VEGF-A, PDGF-B PATHWAY & ANG-2

VEGF-A is also referred to as VEGF, Vascular Endothelial Growth FactorA, MVCD1, VEGF-A, Vascular Endothelial Growth Factor, VascularPermeability Factor and VPF. Human VEGF-A binds to the human FLT1/VEGFR1and KDR/VEGFR2 receptors. Human isoforms, including VEGF-165 andVEGF-145, have been observed.

Vascular endothelial growth factor (VEGF) is a highly specific mitogenfor vascular endothelial cells. Five VEGF isoforms are generated as aresult of alternative splicing from a single VEGF gene. These isoformsdiffer in their molecular mass and in biological properties such astheir ability to bind to cell-surface heparan-sulfate proteoglycans. Theexpression of VEGF is potentiated in response to hypoxia, by activatedoncogenes, and by a variety of cytokines. VEGF induces endothelial cellproliferation, promotes cell migration, and inhibits apoptosis. In vivoVEGF induces angiogenesis as well as permeabilization of blood vessels,and plays a central role in the regulation of vasculogenesis.Deregulated VEGF expression contributes to the development of solidtumours by promoting tumour angiogenesis and to the etiology of severaladditional diseases that are characterized by abnormal angiogenesis.Consequently, inhibition of VEGF signalling abrogates the development ofa wide variety of tumours. The various VEGF forms bind to twotyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1) inhumans, which are expressed almost exclusively in endothelial cells.Endothelial cells express in addition the neuropilin-1 and neuropilin-2coreceptors, which bind selectively to the 165 amino acid form of VEGF(VEGF165).

VEGF is a key regulator of angiogenesis, and its expression in producingcells is regulated by a plethora of external factors. Cytokines, growthfactors, and gonadotropins that do not stimulate angiogenesis directlycan modulate angiogenesis by modulating VEGF expression in specific celltypes, and thus exert an indirect angiogenic or anti-angiogenic effect.Factors that can potentiate VEGF production include fibroblast growthfactor 4 (FGF-4), PDGF, tumour necrosis factor α, transforming growthfactor β (TGF-β), keratinocyte growth factor (KGF), IGF-I, interleukin1β (IL-1β), and IL-6.

Angiogenesis has a causal role in many diseases, including neovascularage-related macular degeneration (AMD). Identification of key regulatorsof angiogenesis, including vascular endothelial growth factor (VEGF),fibroblast growth factor 2, pigment epithelium-derived growth factor,angiopoietins and extracellular matrix molecules, has facilitated thedevelopment of novel therapeutic agents that target the underlyingpathological angiogenic process. Among these, VEGF serves as a “masterswitch” for many ocular neovascular conditions through its promotion ofendothelial cell proliferation and survival, vascular permeability andocular inflammation. Anti-VEGF agents are now available, such asaflibercept (EYLEA™), bevacizumab (AVASTIN™, ranibizumab (LUCENTIS™) andpegaptanib sodium (MACUGEN™), an aptamer for neovascular AMD. Unlikebevacizumab, which binds all VEGF isoforms, pegaptanib targets onlyVEGF165, the isoform responsible for pathological ocularneovascularization.

Flt-1 (VEGFR-1; Unitprot ID: P17948) is one of the two human receptorsfor vascular endothelial growth factor. The other being KDR (VEGFR-2;Uniprot ID: P35968). The flt-1 protein consists of an external domaincontaining seven immunoglobulin like domains, a transmembrane region anda cytoplasmic region containing a tyrosine kinase domain. In contrast toother members of the receptor tyrosine kinase family, the kinase domainof fit-1 is in two segments with an intervening sequence of ˜70 aminoacids. The biology of the VEGF receptors has been reviewed (Neufeld etal., (1999) FASEB Journal. 13:11-22; Zachary (1998) ExperimentalNephrology. 6:480-487) and the tyrosine phosphorylation sites have beenidentified (Ito et al., (1998) J. Biol. Chem. 273:23410-23418).

It is thought that flt-1 may be important in regulating the tissuearchitecture in developing vasculature while the second VEGF receptor(KDR, VEGFR-2) mediates the mitogenic and angiogenic effects of VEGF inendothelial cells. Evidence to support this theory has come fromknockout studies in mice (Fong et al., (1995) Nature. 376:66-70). VEGFand its receptors are over expressed in many tumour types and blockingof VEGF function inhibits angiogenesis and suppresses growth of tumourswhile over expression of VEGF enhances angiogenesis and tumour growth(Skobe et al., (1997) Nature Medicine 3:1222-1227).

The flt-1 cDNA (EMBL Accession Number X51602, 7680 bp) encodes a matureprotein of 1338 amino acids. The structure of the murine flt-1 gene hasbeen determined (Kondo et al., (1998) Gene 208:297-305) and has beenused to predict the intron/exon boundaries within the human gene. Thepromoter region of the human gene has been characterised (Ikeda et al.,(1996) Growth Factors. 13:151-162; Morishita et al., (1995) J Biol Chem270:27948-27953; EMBL Accession Number D64016,1745 bp). The flt-1 gene,which is organised into thirty exons, has been localised to chromosome13q12 (Rosnet et al. (1993) Oncogene 8:73-179). See EP1130123A2.

Diabetic Retinopathy (DR)

Diabetic retinopathy (DR), a micro-vascular complication of diabetes, isa main cause of blindness in adults. It is well established that DR isdetermined by both genetic and environmental factors. The longerduration of diabetes, poorer control of blood glucose and elevated bloodpressure are the major risk factors in the development of DR. However,genetic factors also play important roles in the pathogenesis of DR.Over the past three decades, the number of people with diabetes mellitushas more than doubled globally, making it one of the most importantpublic health challenges to all nations. DR occurs in 75% of patientswith type 2 diabetes mellitus and almost all patients with type 1diabetes mellitus within 15 years of the manifestation of diabetes.Evidence supports an important role for genetics in determining risk forDR.

VEGF-A has been implicated as a major contributor to the development ofDR. VEGF-A could induce the earliest changes in DR includingleukostasis, blood-retinal barrier breakdown, and macular edema andneovascularization in progression of DR. The VEGF-A gene is located onhuman chromosome 6p21.3 and consists of 8 exons. The genetic variants inthe VEGF-A gene are suggested to influence levels of VEGF-A proteinexpression. To date, many studies have investigated the associationsbetween polymorphisms in the VEGF-A gene in humans and DR. −634G/C(+405G/C or rs2010963) polymorphism in the 5-untranslated region and−2578C/A (−25491/D (in high linkage disequilibrium with −2578C/A) orrs699947) polymorphism in the promoter region of the VEGF-A gene havebeen investigated.

Age-Related Macular Degeneration (AMD)

Age-related macular degeneration (AMD) is a neurodegenerative diseasethat leads to visual impairment and accounts for half of all cases ofregistered blindness in Western individuals older than 65 years of age.There are approximately eight million people in the United States withsymptoms of early or intermediate AMD, of whom approximately one millionwill develop advanced AMD within the next five years. AMD is estimatedto affect about 50 million people worldwide, and an increase in agingpopulations makes AMD a significant public health concern and a majorfocus of research efforts. AMD is a clinically heterogeneous andgenetically complex disease, with multiple environmental and geneticrisk factors involved. While epidemiological studies have linkedcigarette smoking, alcohol consumption, light exposure, diet, drugs, andhigh blood pressure to the risk of AMD, familial aggregation and twinstudies have suggested that genetic variation may also play an importantrole in the disease.

Anti-VEGF therapies are now regarded as the standard of care in thetreatment of neovascular AMD. Two of the most widely used treatments areranibizumab and bevacizumab. Ranibizumab (Lucentis®, Genentech, CA,USA), a US FDA-approved anti-VEGF therapy for AMD, is a recombinant,fragmented, monoclonal antibody that binds to VEGF. On the other hand,bevacizumab (Avastin®, Genentech), although not FDA approved for oculartreatment, is a full-length monoclonal antibody reportedly with similarfunction as that of ranibizumab. Bevacizumab, FDA approved formetastatic colon cancer, has been given as an off-label treatment forocular angiogenic disorders. An individual's genetic variation mayaffect the treatment response in both of these drugs.

Polypoidal Choroidal Vasculopathy (PCV)

A subset of wet AMD, termed polypoidal chorio-vasculopathy (PCV), hasbeen identified as a clinical entity. PCV is important, as it may be themain cause of vision loss from AMD in certain Asian and

black populations. The distinctive clinical features of PCV include oneor more haemorrhagic pigment epithelial detachments (PEDs), extensiveexudation, and disease outside of the macula including the periphery.There is a predilection for peripapillary ‘polyps’. Patients are alsocharacteristically younger.

There is increasing evidence that Asian patients with neovascular AMDhave a variant of AMD, ie, polypoidal choroidal vasculopathy (PCV),which is characterized by polypoidal lesion with inner choroidal vesselabnormality. PCV is more prevalent in Asian subjects, reportedlyaccounting for about 50% of neovascular AMD compared to 10% inEuropeans. Studies have shown that anti-VEGF therapy (ranibizumab) incombination with photodynamic therapy (PDT) was associated with a morefavorable outcome compared to ranibizumab monotherapy.

Retinal Angiomatous Proliferation (RAP)

Another variant of AMD in the spectrum of occult CNV is retinalangiomatous proliferation (RAP), which represents about 12%-15% ofneovascular AMD. It is associated with proliferation of intraretinalcapillaries with retinal anastomosis or CNV.

Diabetic macular edema (DME)

Diabetic macular edema (DME), the most common cause of visual loss insubjects with diabetes, is a separate classification assessedindependently from the DR spectrum, because it can develop at any stageof DR. The pathogenesis of DR and DME is thought to be related to theloss of pericytes, thickening of basement membrane and endothelial cellloss, leading to microaneurysms, blood-retinal barrier breakdown,increase in inflammation and vascular leakage. There are severaltreatment modalities for DME. The goal of laser treatment of DME is toreduce disease progression by targeting areas of leakage on the retina.Intravitreal triamcinolone acetonide (IVTA) has also been shown tosignificantly reduce DME, with maximal action at one week and lasting3-6 months.

Retinal-vein occlusion (RVO)

Retinal-vein occlusion (RVO) is the most common retinal vasculardisorder after diabetic retinopathy in the elderly and is oftenassociated with systemic disorders, such as hypertension,hyperlipidemia, diabetes mellitus and/or arteriosclerotic vasculardisorders. It is classified as branch retinal vein occlusion (BRVO) orcentral retinal vein occlusion (CRVO), depending on the site ofocclusion and further divided into ischemic (non-perfusion) ornon-ischemic (perfusion) RVO, each with differing prognosis andtreatment. Macular edema and retinal neovascularization are the two mostcommon causes of visual impairments; thus, ocular managements with laserphotocoagulation, intravitreal injections of glucocorticoids oranti-VEGF agents, as well as other surgical or systemic therapies, havefocused on these two sequelae.

Retinopathy of Prematurity (ROP)

ROP remains one of the leading causes of childhood blindness. In latestages of ROP, neovascularization of abnormal or pathological vesselsarise, due to retinal immaturity, and lead to retinal traction,detachment, hemorrhage and funnel configuration, eventually resulting inpoor vision. Neovascularization is mainly driven by VEGF, and currently,the recommended treatment for Type-1 ROP is peripheral ablation bylaser.

Angiogenesis & Angiogenesis Inhibitors

Angiogenesis is the formation of new blood vessels. This processinvolves the migration, growth, and differentiation of endothelialcells, which line the inside wall of blood vessels. The process ofangiogenesis is controlled by chemical signals in the body. Thesesignals can stimulate both the repair of damaged blood vessels and theformation of new blood vessels. Other chemical signals, calledangiogenesis inhibitors, interfere with blood vessel formation.Normally, the stimulating and inhibiting effects of these chemicalsignals are balanced so that blood vessels form only when and where theyare needed. Neovascularization is the formation of functionalmicrovascular networks with red blood cell perfusion. Neovascularizationdiffers from angiogenesis in that angiogenesis is mainly characterizedby the protrusion and outgrowth of capillary buds and sprouts frompre-existing blood vessels.

Angiogenesis plays a critical role in the growth and spread of cancer. Ablood supply is necessary for tumours to grow beyond a few millimetersin size. Tumours can cause this blood supply to form by giving offchemical signals that stimulate angiogenesis. Tumours can also stimulatenearby normal cells to produce angiogenesis signalling molecules. Theresulting new blood vessels “feed” growing tumours with oxygen andnutrients, allowing the cancer cells to invade nearby tissue, to movethroughout the body, and to form new colonies of cancer cells, calledmetastases. Because tumours cannot grow beyond a certain size or spreadwithout a blood supply, research is focused on trying to find ways toblock tumour angiogenesis, with the idea that angiogenesis inhibitorswill prevent or slow the growth of cancer. Similarly, ocular diseasesand conditions, such as AMD and DR are associated with angiogenesis, andinhibitors are useful for the treatment or prevention of these too.

Thus, anti-VEGF (ie, VEGF-A) ligands and antibodies of the invention areuseful for treating, preventing or reducing the risk of one or more ofsuch ocular conditions and cancer (eg, solid tumours).

Angiogenesis requires the binding of signalling molecules, such asvascular endothelial growth factor (VEGF), to receptors on the surfaceof normal endothelial cells. When VEGF and other endothelial growthfactors bind to their receptors on endothelial cells, signals withinthese cells are initiated that promote the growth and survival of newblood vessels. Angiogenesis inhibitors interfere with various steps inthis process. For example, bevacizumab (Avastin®) is an anti-VEGFmonoclonal antibody that specifically recognizes and binds to VEGF.Aflibercept (Eylea®) is a VEGF receptor-Fc fusion that binds VEGF. Otherangiogenesis inhibitors, including sorafenib and sunitinib, bind toreceptors on the surface of endothelial cells or to other proteins inthe downstream signalling pathways, blocking their activities.

In addition to, or alternative to, targeting human VEGF-A to addressocular conditions or cancer (or angiogenesis or neovascularisationgenerally), it may be useful to target the platelet derived growthfactor pathway (eg, targeting PDGF-B and/or PDGFR-B), or theangiopoietin-2 pathway. Further details are provided below.

In an example, the invention provides a method of treating or reducingthe risk of a VEGF-A-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human VEGF-Aprotein. The invention also provides a corresponding ligand.

In an example, the invention provides a method of treating or reducingthe risk of a PDGF-B (ie, platelet derived growth factor-beta)-mediateddisease or condition in a human in need thereof, the method comprisingadministering to said human a ligand (eg, an antibody or antibodyfragment) that specifically binds a human PDGF-B protein. The inventionalso provides a corresponding ligand.

The present invention provides anti-VEGF-A ligands; and VEGF-A-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of VEGF-A, in particular human VEGF-A or its ligands and inscreening assays to identify other antagonists of VEGF-A activity. Someof the ligands of the invention are useful for inhibitingVEGF-A-mediated activities.

In an example, the invention provides a method of treating or reducingthe risk of a PDGFR-B (ie, platelet derived growth factor-betareceptor)-mediated disease or condition in a human in need thereof, themethod comprising administering to said human a ligand (eg, an antibodyor antibody fragment) that specifically binds a human PDGFR-B protein.The invention also provides a corresponding ligand.

The present invention provides anti-PDGF-B ligands; and PDGF-B-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PDGF-B, in particular human PDGF-B or its ligands and inscreening assays to identify other antagonists of PDGF-B activity. Someof the ligands of the invention are useful for inhibitingPDGF-B-mediated activities.

The present invention provides anti-VEGF-A ligands; and VEGF-A-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of VEGF-A, in particular human VEGF-A or its ligands and inscreening assays to identify other antagonists of VEGF-A activity. Someof the ligands of the invention are useful for inhibitingVEGF-A-mediated activities.

The present invention provides anti-PDGFR-B ligands; and PDGFR-B-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PDGFR-B, in particular human PDGFR-B or its ligands andin screening assays to identify other antagonists of PDGFR-B activity.Some of the ligands of the invention are useful for inhibitingPDGFR-B-mediated activities.

Anti-VEGF-A ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human VEGF-A, which is a commonly-occurring form. While such therapiesare useful for human patients harbouring this form of human VEGF-A, theinventor considered it useful to investigate the possibility oftargeting rarer—but still naturally-occurring—forms of VEGF-A amongsthuman populations. In this way, the inventor arrived at insight into thenatural occurrences and distributions of rarer human VEGF-A forms thatcan serve as useful targets (at the protein or nucleic acid level) forhuman treatment, prophylaxis and diagnosis pertinent to diseases andconditions mediated or associated with VEGF-A activity. Thisparticularly provides for tailored therapies, prophylaxis and diagnosisin humans that are devoid of the common VEGF-A gene or protein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer VEGF-A polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous (aka“missense”) changes at the nucleotide level that translate into one ormore corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention. In particular, humanVEGF-A variations are correlated with increased incidence or risk ofVEGF-A mediated diseases or conditions, such as ocular conditions orcancer. Additionally or alternatively, VEGF-A variations are correlatedwith improved responses following treatments for ocular conditions. Theinventor analysed such variations and devised the collections ofvariants in Tables 18-20. As shown in those tables, the inventor alsoassessed variation in other human targets that also correlate toincidence or risk of diseases or conditions, such as ocular conditionsor cancer, or improved responses following treatments for ocularconditions. In this respect, therefore, the invention provides variousaspects and concepts as set out below in this example.

The inventor saw that there is significant industrial and medicalapplication for the invention in terms of guiding the choice of anti-TOI(VEGF-A, PDGF-B and/or PDGFR-B) ligand for administration to humanpatients for therapy and/or prophylaxis of VEGF-A, PDGF-B and/orPDGFR-B-mediated or associated diseases or conditions. In this way, thepatient receives drugs and ligands that are tailored to their needs—asdetermined by the patient's genetic or phenotypic makeup. Hand-in-handwith this, the invention provides for the genotyping and/or phenotypingof patients in connection with such treatment, thereby allowing a propermatch of drug to patient. This increases the chances of medicalefficacy, reduces the likelihood of inferior treatment using drugs orligands that are not matched to the patient (eg, poor efficacy and/orside-effects) and avoids pharmaceutical mis-prescription and waste.

In developing this thinking, in a non-limiting embodiment the presentinventor decided to determine a set of human VEGF-A, PDGF-B and PDGFR-Bvariants on the basis of the following criteria, these being criteriathat the inventor realised would provide for useful medical drugs anddiagnostics to tailored need in the human population. The inventorselected variants having at least 3 of the 4 following criteria:—

Naturally-occurring human TOI (VEGF-A, PDGF-B or PDGFR-B) variationhaving a cumulative human allele frequency of 35% or less;

Naturally-occurring human TOI variation having a total human genotypefrequency of about 50% or less;

Naturally-occurring human TOI variation found in many different humanethnic populations (using the standard categorisation of the 1000Genomes Project; see Table 2 below); and

Naturally-occurring human TOI variation found in many individualsdistributed across such many different ethnic populations.

On the basis of these criteria, the inventor identified variants listedbelow.

Furthermore, the inventor identified useful variations also in thefollowing human TOIs: complement factor H (CFH), complement component 2(C2), prothrombin, methylenetetrahydrofolate reductase (MTHFR), FactorV, ARMS2, VEGFR2 and HTRA1. As the titles to Tables 18-20 indicate, theexaminer realised that such variants have useful correlations.

In an example, the ligand of the invention comprises an anti-TOI (eg,anti-human VEGF-A) binding site, wherein the binding site is a human orhumanized binding site, eg, the binding site comprises or consists of ahuman or humanized antibody variable domain or plurality of variabledomains (eg, human VH/VL binding site(s)). Additionally oralternatively, the ligand comprises one or more human antibody constantregions (eg, a human antibody CHL CH2, CH3 (or all of these) or Fc). Inan example, the ligand is an antibody that comprises human or humanizedvariable regions and human constant regions (eg, bearing one or moremutations to enhance or dampen Fc function in a human patient).

In an example, the invention provides a method of targeting VEGF-A in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human VEGF-Aprotein that is encoded by a human VEGF-A nucleotide comprising a VEGF-ASNP as described herein. In an example, the human is suffering from orat risk of a VEGF-A-mediated disease or condition. In an example, themethod treats or reduces the risk of a VEGF-A-mediated disease orcondition in the human. In an example, the condition comprisesangiogenesis or neovascularisation.

In an example, the invention provides a method of targeting PDGF-B in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human PDGF-Bprotein that is encoded by a human PDGF-B nucleotide comprising a PDGF-BSNP as described herein. In an example, the human is suffering from orat risk of a PDGF-B-mediated disease or condition. In an example, themethod treats or reduces the risk of a PDGF-B-mediated disease orcondition in the human. In an example, the condition comprisesangiogenesis or neovascularisation.

In an example, the invention provides a method of targeting PDGFR-B in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a humanPDGFR-B protein that is encoded by a human PDGFR-B nucleotide comprisinga PDGFR-B SNP as described herein. In an example, the human is sufferingfrom or at risk of a PDGFR-B-mediated disease or condition. In anexample, the method treats or reduces the risk of a PDGFR-B-mediateddisease or condition in the human. In an example, the conditioncomprises angiogenesis or neovascularisation.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40; andwherein (ii) said human comprises said nucleotide sequence encoding saidVEGF-A, PDGF-B or PDGFR-B.

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises said nucleotide sequence encoding said VEGF-A, PDGF-B orPDGFR-B.

In a specific embodiment, the anti-VEGF-A, PDGF-B or PDGFR-B ligand,antibody or fragment of present invention comprises an Fc region,wherein the Fc region comprises at least one non-native amino acidresidue selected from the group consisting of 234D, 234E, 234N, 234Q,234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S,235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N,239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y,241E, 241R. 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G,251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 2631, 263A,263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G,265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 266I, 266A, 266T,266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P,292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S,297D, 297E, 298H, 2981, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H,299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T,325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q,328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 3301,330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M,331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R,331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H,332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Yand 434W as numbered by the EU index as set forth in Kabat. Optionally,the Fc region may comprise additional and/or alternative non-nativeamino acid residues known to one skilled in the art (see, e.g., U.S.Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO05/040217).

The ligand, antibody or fragment according to the invention (eg, ananti-VEGF-A ligand, antibody or fragment) is for treating or preventingor reducing the risk of (or treats or prevents or reduces the risk of),for example, any disease or condition disclosed in US2012064621A1 orU.S. Pat. No. 7,704,500B2, the disclosure of which diseases andconditions are incorporated herein by reference for potential inclusionin one or more claims herein. Guidance on obtaining and testingantibodies can also be found in that PCT application. Suitableanti-VEGF-A ligands, antibodies or fragments for use in the inventionare disclosed in US2012064621A1 or U.S. Pat. No. 7,704,500B2, thedisclosure of which (including sequences thereof) are incorporatedherein by reference for potential inclusion in one or more claimsherein.

Further encompassed by the invention is the use of the anti-TOI (eg,VEGF-A) ligand, antibody or fragment in the manufacture of a medicamentfor use to attenuate or inhibit a TOI (eg, VEGF-A)-mediated disease ordisorder in a human. VEGF-A-mediated or related disorders which aretreated by the ligand, antibody or fragment of the invention include,for example, described below.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving TOI (eg,VEGF-A) expression and/or activity. One embodiment, among others, is amethod of treatment comprising administering an effective amount of aligand, antibody or fragment of the invention to a patient in needthereof, wherein functional consequences of TOI activation aredecreased. Another embodiment, among others, is a method of treatmentcomprising (i) identifying a patient demonstrating TOI expression oractivity, and (ii) administering an effective amount of a ligand,antibody or fragment of the invention to the patient, wherein afunctional consequence of TOT activation are attenuated. An effectiveamount according to the invention is an amount that modulates (e.g.decreases) the functional consequences of TOI activation so as tomodulate (e.g. decrease or lessen) the severity of at least one symptomof the particular disease or disorder being treated, but not necessarilycure the disease or disorder. Accordingly, one embodiment of theinvention is a method of treating or reducing the severity of at leastone symptom of any of the disorders referred to herein, comprisingadministering to a patient in need thereof an effective amount of one ormore ligands, antibodies or fragments of the present invention alone orin a combined therapeutic regimen with another appropriate medicamentknown in the art or described herein such that the severity of at leastone symptom of any of the disorders is reduced. Another embodiment ofthe invention, among others, is a method of antagonizing at least oneeffect of TOI comprising contacting with or administering an effectiveamount of one or more ligands, antibodies or fragments of the presentinvention such that said at least one effect of TOI is antagonized, e.g.the ability to promote or maintain angiogenesis or neovascularisation.

Tailoring Antibodies to Rarer TOI (VEGF-A, PDGF-B or PDGFR-B) VariantProfile

As outlined herein (for example, in the context of PCSK9 in Example 1),the invention includes the possibility to tailor treatment of humansfurther by selecting antibody-based ligands with variable domains and/orconstant domains based on gene segments found in many humans of theethnic populations where the variant TOI forms are found to meet theselection criteria of the invention. This also applies mutatis mutandiswhere the TOI is human VEGF-A, PDGF-B or PDGFR-B as in the presentexample. Thus, all disclosure herein relating to tailoring variableand/or constant domains apply to the present example, relating toVEGF-A, PDGF-B or PDGFR-B and is combinable for use in one or moreclaims herein.

As described in Example 1, an example is provided for ligands comprisingantibody VH domains derived from recombination of human IGHV genesegments comprising selected nucleotides at positions in the HCDR1 orFW3 where there is variability in humans (ie, where SNPs occur inhumans).

Further information is provided in Table 4, which shows variation atthese positions, as well as the variant distributions across the 1000Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ or constant region gene segments (see further Table 9 forrepresentative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4.(vi) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Valat position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shownin SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asnat position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ IDNO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257shown in SEQ ID NO: 6.(vii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(viii) wherein the ligand comprises a human IGLC1*01 lambda chainconstant region and wherein said human comprises (i) a human IGLC1*01lambda chain constant region gene segment, or the human expressesantibodies comprising human IGLC1*01 lambda chain constant regions.(ix) wherein the ligand comprises a human gamma-4 heavy chain constantregion that comprises a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73 and wherein said humancomprises (i) an IGHG4*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-4heavy chain constant regions comprising a Leu at position 189 shown inSEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73.(x) wherein the ligand comprises a human gamma-3 heavy chain constantregion encoded by a first human IGHG3 (eg, IGHG3*01) constant regiongene segment and wherein said human comprises (i) said first constantregion gene segment (eg, an IGHG3*01), or the human expresses antibodiescomprising human gamma-3 heavy chain constant regions encoded by saidfirst human IGHG3 (eg, IGHG3*01) constant region gene segment.(xi) wherein the ligand comprises a human epsilon heavy chain constantregion encoded by a first human epsilon heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human epsilonheavy chain constant regions encoded by said first constant region genesegment.(xii) wherein the ligand comprises a human mu heavy chain constantregion encoded by a first human mu heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human muheavy chain constant regions encoded by said first constant region genesegment.(xiii) wherein the ligand comprises a human alpha heavy chain constantregion encoded by a first human alpha heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human alphaheavy chain constant regions encoded by said first constant region genesegment.(xiv) wherein the ligand comprises a human delta heavy chain constantregion encoded by a first human delta heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human deltaheavy chain constant regions encoded by said first constant region genesegment.(xv) wherein the ligand comprises a human kappa light chain constantregion encoded by a first human kappa light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human kappalight chain constant regions encoded by said first constant region genesegment.(xvi) wherein the ligand comprises a human lambda light chain constantregion encoded by a first human lambda light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human lambdalight chain constant regions encoded by said first constant region genesegment.

It may be advantageous for VEGF-A, PDGF-B or PDGFR-B ligands, for theligand to comprise a gamma-1 or gamma-2 constant region (eg, as per theembodiments (i) to (xvi) above). For example, the ligand comprises saidgamma-1 constant region, eg, an ADCC or CDC enhanced constant regionmeeting embodiment (v).

Determination of Specific Binding of Ligands of the Invention to VEGF-A,PDGF-B or PDGFR-B Variants

The specific binding of ligands of the invention to VEGF-A, PDGF-B orPDGFR-B variants can be performed using the SPR method described inExample 1.

Human Polymorphisms References

-   1. PLoS One. 2013 Dec. 20; 8(12):e84069. doi:    10.1371/journal.pone.0084069, eCollection 2013, “Association of VEGF    gene polymorphisms with diabetic retinopathy: a meta-analysis”, Gong    J Y et al. The authors reported a meta analysis which, in    conclusion, reportedly demonstrated that DR is associated with VEGF    gene −460T/C polymorphism (rs833061).-   2. J Diabetes Res. 2014; 2014:805801. doi: 10.1155/2014/805801, Epub    2014 Apr. 28, “The associations between VEGF gene polymorphisms and    diabetic retinopathy susceptibility: a meta-analysis of 11    case-control studies”, Han L et al. A total of 11 studies fulfilling    the inclusion criteria were included in this meta-analysis. A    significant relationship between VEGF+936C/T (rs3025039)    polymorphism and DR was reported.-   3. BMC Ophthalmol. 2013 Oct. 16; 13:56. doi:    10.1186/1471-2415-13-56, “Two polymorphisms (rs699947, rs2010963) in    the VEGF-A gene and diabetic retinopathy: an updated meta-analysis”,    Lu Y et al. The authors reported meta-analysis confirmed the    significant association between rs699947 polymorphism and DR after    exclusion of outliers.-   4. Gene. 2013 Apr. 15; 518(2):310-5. doi:    10.1016/j.gene.2013.01.018. Epub 2013 Jan. 24. VEGF −634G>C    polymorphism and diabetic retinopathy risk: a meta-analysis.    -   Qiu M et al. The aim of the published study was to investigate        whether VEGF −634G>C polymorphism is associated with the risk of        DR in type 2 diabetes. A systematic search of electronic        databases (PubMed, Embase and Web of Science) and reference        lists of relevant articles was carried out until Sep. 15, 2012.        The pooled odds ratios (ORs) and their corresponding 95%        confidence interval (CI) were calculated by a fixed effect        model. A total of 1525 DR cases and 1422 diabetic without        retinopathy (DWR) controls in 9 independent studies were        included in the meta-analysis. A significant relationship        between VEGF −634G>C polymorphism and DR was found in an allelic        genetic model (OR: 1.13, 95% CI: 1.01 to 1.25, P=0.03) and a        recessive genetic model (OR: 1.26, 95% CI: 1.02 to 1.55,        P=0.03). The authors concluded that their research confirmed the        association between the VEGF −634G>C polymorphism and DR in        subjects with type 2 diabetes.-   5. Diabetes. 2002 May; 51(5):1635-9, “A common polymorphism in the    5′-untranslated region of the VEGF gene is associated with diabetic    retinopathy in type 2 diabetes”, Awata T et al. These authors    suggest that the −634G>C polymorphism in the 5′UTR of the VEGF gene    is a genetic risk factor for diabetic retinopathy.-   6. Molecular Vision 2010;    16:1958-1981<http://www.molvis.org/molvis/v16/a213>, Tong Y et al.    In conclusion, according to the authors the described Human Genome    Epidemiology (HuGE) systematic review presents strong evidence for    an association between the HTRA1 rs11200638 G→A polymorphism,    LOC387715/ARMS2rs10490924 G→T polymorphism, and AMD, and suggests    that both of these genes play important roles in this disease.    Potential gene-gene and gene-environmental interactions and possible    mechanisms of AMD are also summarized and discussed.-   7. J. Pers. Med. 2013, 3, 40-69; doi:10.3390/jpm3010040,    “Personalized Medicine in Ophthalmology: From Pharmacogenetic    Biomarkers to Therapeutic and Dosage Optimization”, Frank S Ong et    al. This review comments that: In the case of intravitreal    bevacizumab, CFH Y402H genotypes, TC and TT, show more than    five-fold increased improvement compared to the CC genotype. The    data shows that after treatment with bevacizumab, visual acuity of    the patients improved from 20/248 to 20/166 (TT) and from 20/206 to    20/170 (TC), but actually decreased from 20/206 to 20/341 for the CC    genotype (p=0.016). In a prospective study with twice the number of    patients, the CC genotype was confirmed to have worse outcome as    measured by distance and reading visual acuity. In a similar    experiment with intravitreal ranibizumab, the TC and TT genotypes    for CFH showed improvement with fewer injections compared to the CC    genotype. Recurrent analysis showed that patients homozygous for the    CFHY 402H risk allele (CC) were 37% more likely to require    additional ranibizumab injections (p=0.04). Another study found that    individuals homozygous for 69S in ARMS2 had decreased central    subfield retinal thickness and no improvement in visual outcomes    compared to improved visual acuity in ARMS2 rs10490924 and rs1061170    genotypes following ranibizumab treatment. Taking genetics and    environmental factors together, the CFH Y402H homozygous CC genotype    with BMI≧30 and smoking reportedly conferred the greatest risk.-   8. Expert Rev Ophthalmol. 2013 Apr. 1; 8(2): 127-140.    doi:10.1586/eop.13.3, “Genetic risk, ethnic variations and    pharmacogenetic biomarkers in age-related macular degeneration and    polypoidal choroidal vasculopathy”, Jane Z. Kuo et al. This article    comments that: Variants in the complement factor H (CFH) gene    confers a 7.4-fold increased likelihood of late AMD. AMD is a common    complex disorder with complex inheritance patterns due to various    gene and environmental interactions. Yet, despite these challenges,    AMD represents an unusual example of the “common disease common    variant” theory in which several variants identified on the CFH gene    have relatively large effects on disease outcome. Even though the    most well-established and consistent association of AMD is observed    with complement factor H (CFH) on chromosome 1, recent studies have    also shown that other candidate genes in the complement pathway,    ARMS2/HTRA1 on chromosome 10q26, LIPC, CETP, and TIMP2 are also    susceptible loci associated with AMD. In 2005, three independent    research groups identified a common coding variant, Y402H    (rs1061170), in the CFH gene on chromosome 1 that was strongly    associated with susceptibility of AMD, with reported odds ratio (OR)    ranging from 2.5 to 3.3 for all AMD and as high as 7.4 for advanced    AMD. The Y402H variant results in a tyrosine-to-histidine    substitution. The association of CFH in AMD implicated that the    complement cascade plays a critical role in the development of AMD    and is now also regarded as a major contributor to susceptibility of    AMD. In addition to CFH and the complement pathway, the region at    chromosome 10q26 also harbors additional signals to AMD    susceptibility, namely the age-related maculopathy susceptibility 2    (ARMS2) and the high-temperature requirement factor H (HTRA1) genes.    It is also possible that susceptible variants within ARMS2 modulate    the promoter activity of HTRA1, so that both ARMS2/HTRA1 contribute    to AMD susceptibility. In one European study, the SNP rs11200638 at    the promoter region of HTRA1 confers a 49.3% attributable risk and    the risk allele was associated with an elevated mRNA and protein    expression and level of HTRA1. The risk allele of the same SNP was    highly significant in the Chinese and has 10 times the risk of    developing AMD compared to subjects with the wild-type allele. Two    VEGF polymorphisms, rs699947 and rs2146323, were significantly    associated with PDT treatment response. The allele frequency of    rs699947 for AA, AC, and CC were 14%, 39%, and 46% in non-responders    versus 40%, 48%, and 12% in responders, respectively (p=0.0008). The    allele frequency of rs2146323 for AA, AC, and CC were 4%, 32%, and    64% in non-responders versus 24%, 38%, and 38% in responders,    respectively (p=0.0369). Thus, the C allele in both SNPs in VEGF    were associated with a higher rate of non-responder to PDT (p=0.0003    for rs699947 and p=0.0036 for rs2146323). In classic choroidal    neovascularization (CNV), prothrombin (G20210A) and MTHFR (C677A)    were significantly associated with PDT responders. In occult CNV,    prothrombin (G20210A) and factor V (G1691A) were significantly    associated with PDT responders. Two studies have showed a worse    visual improvement in subjects with the homozygous TT risk allele at    ARMS2 rs10490924 (A69S) after ranibizumab or bevacizumab injections.    Other candidate gene studies found that patients with the risk    genotype (CC) at rs1413711 of VEGF has a more significant visual    gain after ranibizumab, the G allele at rs699946 of VEGF was    significantly associated with a better visual prognosis after either    intravitreal bevacizumab or triple therapy (PDT with intravitreal    bevacizumab and triamcinolone acetonide). In a study examining the    cumulative effect of risk alleles at CFH (Y402H; rs1061170), ARMS2    (A69S; rs10490924), VEGF (rs699947 and rs833069), KDR (rs2071559 and    rs7671745), LPR5 (rs3736228), and FZD4 (rs10898563) after    ranibizumab treatment, subjects without the high-risk alleles in CFH    and ARMS2 have significantly more visual improvement compared to    subjects with the high-risk alleles (p=0.009). The mean visual    acuity improvement was 10 letters in subjects without the 4-risk    alleles, compared to no improvement in subjects with all 4-risk    alleles. By adding VEGF into the model, they found that subjects    with all 6-risk alleles in CFH, ARMS2, and VEGF demonstrated a mean    loss of 10 letters after ranibizumab treatment, whereas the other    allele groups all demonstrated improvement (p<0.0001), suggesting a    cumulative effect of the risk alleles with a poorer response rate to    treatment. A comprehensive meta-analysis found variants at    ARMS2/HTRA1 (rs10490924, OR=2.27, p<0.00001; rs11200638, OR=2.72,    p<0.00001), CFH (rs1061170, OR=1.72, p<0.00001; rs800292, OR=2.10,    p<0.00001) or the complement pathway (C2; rs547154, OR=0.56, p=0.01)    were significantly associated with PCV, with higher odds ratio    observed at variants in ARMS2/HTRA1. The strong association of    ARMS2/HTRA1 to PCV was supported by numerous studies of Asian    populations.-   9. Survey of Ophthalmology, Volume 59, Issue 1, January-February    2014, Pages 1-18, “Predictors of anti-VEGF treatment response in    neovascular age-related macular degeneration”, Finger et al. The    authors reviewed evidence on predictors of anti-vascular endothelial    growth factor (VEGF) treatment response in neovascular age-related    macular degeneration. Just under half of the SNPs assessed in the    CFH gene and 15% of the SNPs assessed in the VEGF gene were found to    be associated with visual outcomes or the number of injections    required during follow-up.-   10. Ophthalmology. 2013 January; 120(1):115-21. doi:    10.1016/j.ophtha.2012.10.006. Epub 2012 Nov. 11, “Variants in the    VEGFA gene and treatment outcome after anti-VEGF treatment for    neovascular age-related macular degeneration2, Abedi F et al. The    authors concluded that: Pharmacogenetic association with anti-VEGF    treatments may influence the visual outcomes in neovascular AMD. In    patients with the T allele in tSNP rs3025000, there was a    significantly better visual outcome at 6 months and a greater chance    of the patients belonging to the responder group with anti-VEGF    treatment at 3, 6, and 12 months. The visual acuity (VA) outcomes of    patients harbouring the T allele at SNP rs3025000 were comparable    with those of the pivotal clinical trials but with fewer injections.-   11. Ophthalmology. 2014 April; 121(4):905-10. doi:    10.1016/j.ophtha.2013.10.047. Epub 2013 Dec. 21, “Polymorphisms in    vascular endothelial growth factor receptor 2 are associated with    better response rates to ranibizumab treatment in age-related    macular degeneration”, Hermann M M et al. The authors evaluated the    association of single nucleotide polymorphisms (SNPs) in VEGF genes    and their receptors (VEGFR) with the response rate to ranibizumab in    366 patients with neovascular AMD. Univariate analyses of variance    (ANOVAs) revealed a significant effect of SNP rs4576072 in the    VEGFR2 gene on VA change after 12 months (F[1,235]=14.05; P=0.02). A    stepwise linear regression analysis returned a model (P=0.01) with    SNPs rs4576072 and rs6828477 in the VEGFR2 gene as independent    predictors for VA change after 12 months, with a mean increase in VA    of 0.26 on the logarithm of the minimum angle of resolution (log    MAR) scale in patients with 3 contributing minor alleles compared    with a loss of 0.03 log MAR in patients with no minor allele.-   12. Research and Reports in Neonatology 2011:1 5-11; “VEGF 936C>T is    predictive of threshold retinopathy of prematurity in Japanese    infants with a 30-week gestational age or less”, Yagi M et al. The    authors found that VEGF 936C>T polymorphism and duration of oxygen    administration were independent risk factors for threshold ROP using    a multivariate logistic analysis with adjustment for gestational age    and birth weight.-   13. Clin Cancer Res 2009; 15(17) Sep. 1, 2009, pp 5297-5302, DOI:    10.115811078-0432.CCR-08-2576, “The Role of Vascular Endothelial    Growth Factor Genetic Variability in Cancer”, Bryan P. Schneider et    al. The authors comment that recently, genetic variability in VEGF    has been studied as a potential predictive biomarker for    bevacizumab. The VEGF-1154 AA and -2578 AA genotypes predicted an    improved median overall survival, whereas the VEGF-634 CC and -1498    TT genotypes predicted protection from grade 3-4 hypertension in the    pivotal trial, E2100. If validated, these finding could help direct    which subgroup of patients should receive bevacizumab.

The inventor identified the following SNPs as SNPs of interest for thepresent invention.

TABLE 18 POLYMORPHISM RISK FACTORS FOR OCULAR CONDITIONS SNP LOCATIONASSOCIATED (forward EXAMPLE POLYMORPHISM SNP ID strand) INDICATIONVEGF-A −2578C > A rs699947  6: 43768652 DR −460T > C rs833061  6:43769749 DR −634G > C rs2010963  6: 43770613 DR +936C > T rs3025039  6:43784799 DR ROP A > G rs699946  6: 43764932 DR HTRA1 −625G > Ars11200638 10: 122461028 AMD ARMS2 G > T rs10490924 10: 122454932 AMDA69S PCV COMPLEMENT FACTOR H (CFH) 184G > A rs800292  1: 196673103 AMDV62I PCV COMPLEMENT COMPONENT 2 (C2) 62G > T rs547154  6: 31943161 AMDPCV

NB: as is known in the art SNP location is expressed as chromosomenumber:nucleotide position, eg, 6:43768652 is nucleotide number 43768652on human chromosome 6. ARMS2 is also known as age-related maculopathysusceptibility protein 2, ARMD8 and ARMS2 HUMAN LOC387715.

TABLE 19 POLYMORPHISMS ASSOCIATED WITH IMPROVED OCULAR RESPONSES SNPLOCATION ASSOCIATED (forward EXAMPLE POLYMORPHISM SNP ID strand) BENEFITVEGF-A −2578C > A rs699947  6: 43768652 AA and AC genotypes associatedwith improved PDT response −111C > A rs2146323  6: 43777358 AA and ACgenotypes associated with improved PDT response −1400T > C rs1413711  6:43772941 CC genotype shows more significant visual gain after anti- VEGFtreatment A > G rs699946  6: 43764932 G significantly associated withbetter visual prognosis after anti-VEGF treatment 398G > A rs833068  6:43774790 AA or GG genotype associated with less anti-VEGF treatmentinjections needed 450T > C rs833069  6: 43774842 AA (TT) or GG (CC)genotype associated with less anti-VEGF treatment injections needed−28C > T rs3025000  6: 43778432 T allele associated with visual acuityimprovement with lower anti- VEGF administrations COMPLEMENT FACTOR H(CFH) T > C rs1061170  1: 196690107 TC and TT Y402H genotypes: Improvedvisual acuity following anti-VEGF treatment −2362T > C rs3766404  1:196682702 TT (AA) genotype associated with less anti-VEGF treatmentinjections needed prothrombin 20210G > A rs1799963 11: 46739505Associated with improved PDT response Methylenetetrahydrofolatereductase (MTHR) 677C > A rs1801133  1: 11796321 Associated with A222Vimproved PDT response FACTOR V 1691G > A rs6025  1: 169549811 Associatedwith R506Q improved PDT response ARMS2 G > T rs10490924 10: 122454932 GGgenotype A69S associated with improved anti- VEGF response VEGFR21026A > G rs4576072  4: 55120071 G allele associated with better visualacuity following anti-VEGF 1263G > A rs6828477  4: 55100634 A alleleassociated with better visual acuity following anti-VEGF

TABLE 20 POLYMORPHISMS ASSOCIATED WITH IMPROVED CANCER RESPONSES SNPLOCATION ASSOCIATED (forward EXAMPLE POLYMORPHISM SNP ID strand) BENEFITVEGF-A −2578C > A rs699947 6: 43768652 AA genotype associated withimproved breast cancer response to anti- VEGF −1154G > A rs1570360 6:43770093 AA genotype associated with improved breast cancer response toanti- VEGF

The invention thus provides the following concepts:—

Human VEGF-A SNPs Associated With VEGF-A Mediated Diseases & Conditions

1. A method of treating or reducing the risk of a disease or conditionmediated by VEGF-A in a human, the method comprising administering tosaid human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody orantibody fragment) that specifically binds to a human VEGF-A that isexpressed by a VEGF-A nucleotide sequence comprising a SNP selected fromthe group consisting of rs699947, rs833061, rs2010963, rs3025039,rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 andrs1570360, wherein said human comprises a VEGF-A nucleotide sequencecomprising said selected SNP.Herein, specific binding can be tested using a sample VEGF-A using aroutine method known the skilled person, eg, ELISA or SPR (eg, using amethod as described herein). For example, the VEGF-A is provided by asample from said human or another human, eg, a serum or blood sample.In an example, the ligand comprises an antibody constant region (eg, anantibody Fc region). Optionally, the ligand comprises a human gamma-1heavy chain constant region that comprises an amino acid selected fromthe group consisting of an Asp corresponding to position 204 of SEQ IDNO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 andwherein said human comprises an IGHG1*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-1 heavy chain constant regions comprising said selected aminoacid. Optionally, this constant region is comprised by an Fc region ofthe ligand.In an example, the ligand comprises an anti-VEGF-A binding site thatspecifically binds human VEGF-A. Optionally, the binding site comprisesone or more antibody variable domains or one or more human VEGF-Areceptor domains. In an example, specific binding with a Kd of 1 mm orless (ie, 1 mM or stronger binding), eg, 1 nM or less; or 100 pM orless; or 10 pM or less. Specific binding can be determined, for example,by assessing binding of the ligand to VEGF-A in a sample from said humanor another human comprising said VEGF-A. In an example, the samplecomprises serum, blood, faeces, tissue, a cell, urine and/or saliva ofsaid human.Optionally, the VEGF-A is a VEGF-A-165 isoform.In an example, the ligand is an anti-VEGF-A trap, eg, aflibercept. In anexample, the ligand is EYLEA™In an example, the ligand is an anti-VEGF-A antibody or antibodyfragment, eg, ESBA 1008, bevacizumab or ranizumab. In an example, theligand is AVASTIN™ or LUCENTIS™.In an example, the ligand is an anti-VEGF ankyrin repeat protein(DARPin), eg, abicipar pegol.In an example, the ligand is an NCE (so-called New Chemical Entity inthe art), eg, sunitinib.In an example, the condition is selected from the group consisting ofangiogenesis, neovascularisation, ocular vascularisation and solidtumour vascularisation.In an example, the condition is subretinal neovascularisation.In an example, the disease or condition is an angiogenic disease orcondition.Optionally the disease or condition is an angiogenic ocular condition orangiogenic cancer, eg a solid tumour, a gastrointestinal cancer,colorectal cancer, liver cancer, breast cancer, ovarian cancer, nonsmall cell lung cancer, thyroid cancer, an oral cancer, or anhaematological cancer.Optionally the angiogenic disease or condition is selected from thegroup consisting of psoriasis, rheumatoid arthritis, a hemangioma, anangiofibroma, diabetic retinopathy, corneal neovacularisation andneovascular glaucoma.Optionally the disease or condition is or comprises vascularpermeability (eg, ocular vascular permeability), edema or inflammationin said human.For example, the disease or condition is selected from the groupconsisting of brain edema (eg, edema associated with brain injury,stroke or brain tumour); edema associated with an inflammatory disorder(eg, associated with psoriasis or arthritis, rheumatoid arthritis orasthma); edema associated with burns; ascites or pleural effusion (eg,associated with tumours, inflammation or trauma); chronic airwayinflammation; capillary leak syndrome; sepsis; kidney disease (eg,associated with increased leakage of protein); and an eye disorder (eg,age related macular degeneration or diabetic retinopathy).In an example the method is for reducing or reducing the risk of VEGF-Amediated endothelial cell maintenance or proliferation in an angiogenicdisease or condition in said human.In an example, the angiogenic disease or condition is an angiogenicdisease or condition recited above, eg, an angiogenic ocular condition.In an example the method is for regressing neovascularisation in aVEGF-A mediated disease or condition in said human.In an example the method is for treating or reducing the risk ofneovascularisation in a human, wherein the human has VEGF-Aligand-refractory neovascularisation.In an example, the human has been diagnosed with said disease orcondition before said administration of the ligand.For example, the human is partially or completely resistant to treatmentwith an anti-VEGF-A ligand, eg, aflibercept, sunitinib, bevacizumab orranizumab.In an example, the ligand is administered by intravenous or subcutaneousadministration and/or is comprised in an injectable preparation.2. The method of concept 1, wherein the condition is an ocularcondition, eg, diabetic retinopathy or retinopathy of prematurity.In an example, the condition is diabetic retinopathy (DR).In an example, the condition is age-related macular degeneration (AMD),eg, exudative AMD.In an example, the condition is polypoidal choroidal vasculopathy (PCV).In an example, the condition is retinal angiomatous proliferation (RAP).In an example, the condition is diabetic macular edema (DME).In an example, the condition is retinal-vein occlusion (RVO).In an example, the condition is retinopathy of prematurity (ROP).In an alternative, instead of “a method of treating or reducing the riskof a disease or condition mediated by VEGF-A in a human”, the inventionrelates to “a method of improving visual acuity in a human”.3. The method of concept 1 or 2, wherein the SNP is selected from thegroup consisting of rs699947, rs833061, rs2010963, rs3025039 andrs699946.In an example, the SNP is rs699947 and optionally the condition is DR.In an example, the SNP is rs833061 and optionally the condition is DR.In an example, the SNP is rs2010963 and optionally the condition is DR.In an example, the SNP is rs3025039 and optionally the condition is DR.In an example, the SNP is rs699946 and optionally the condition is DR.4. The method of concept 1 or 2, wherein the SNP is selected from thegroup consisting of rs699947, rs2146323, rs1413711, rs699946, rs833068,rs833069 and rs3025000.As shown in Table 19, these SNPs are associated with improved ocularresponses following ocular treatment (eg, photodynamic therapy (PDT) orVEGF-A antagonism).In an example, the SNP is rs699947, eg, homozygous for said SNP.In an example, the SNP is rs2146323, eg, homozygous for said SNP.In an example, the SNP is rs1413711, eg, homozygous for said SNP.In an example, the SNP is rs699946, eg, homozygous for said SNP.In an example, the SNP is rs833068, eg, homozygous for said SNP.In an example, the SNP is rs833069, eg, homozygous for said SNP.In an example, the SNP is rs3025000, eg, homozygous for said SNP.5. The method of concept 3 or 4, wherein the human is homozygous forsaid SNP.In another example, the human is heterozygous for said SNP.6. The method of any one of concepts 2 to 5, wherein the method furthercomprises treating the human with photodynamic therapy.For example, the method further comprises treating the human with PDTsimultaneously with, or after, administration of said ligand to thehuman.7. The method of concept 1, wherein the disease or condition is acancer.8. The method of concept 7, wherein the SNP is rs699947 or rs1570360.In an example, the SNP is rs699947, eg, homozygous for said SNP.In an example, the SNP is rs1570360, eg, homozygous for said SNP.9. The method of any preceding concept, wherein the human comprises (a)a HTRA1 nucleotide sequence comprising SNP rs11200638; (b) an ARMS2nucleotide sequence comprising SNP rs10490924; (c) a CFH nucleotidesequence comprising SNP rs800292, a Tat the position of rs1061170 or anA at the position of rs3766404; (d) a complement component 2 (C2)nucleotide sequence comprising SNP rs547154; (e) a prothrombinnucleotide sequence comprising SNP rs1799963; (f) a MTHFR nucleotidesequence comprising SNP rs1801133; (g) a Factor V nucleotide sequencecomprising SNP rs6025; or (h) a VEGFR2 nucleotide sequence comprisingSNP rs4576072 or rs6828477.In an example the human comprises a HTRA1 nucleotide sequence comprisingSNP rs11200638, eg, in homozygous form.In an example the human comprises an ARMS2 nucleotide sequencecomprising SNP rs10490924, eg, in homozygous form.In an example the human comprises a CFH nucleotide sequence comprisingSNP rs800292, eg, in homozygous form.In an example the human comprises a CFH nucleotide sequence comprisingSNP rs1061170, eg, in homozygous form.In an example the human comprises a CFH nucleotide sequence comprisingSNP rs3766404, eg, in homozygous form.In an example the human comprises a complement component 2 (C2)nucleotide sequence comprising SNP rs547154, eg, in homozygous form.In an example the human comprises a prothrombin nucleotide sequencecomprising SNP rs1799963, eg, in homozygous form.In an example the human comprises a MTHFR nucleotide sequence comprisingSNP rs1801133, eg, in homozygous form.In an example the human comprises a Factor V nucleotide sequencecomprising SNP rs6025, eg, in homozygous form.In an example the human comprises a VEGFR2 nucleotide sequencecomprising SNP rs4576072, eg, in homozygous form.In an example the human comprises a VEGFR2 nucleotide sequencecomprising SNP rs6828477, eg, in homozygous form.10. The method of any one of concepts 2 to 6, wherein the humancomprises (a) a HTRA1 nucleotide sequence comprising SNP rs11200638; (b)an ARMS2 nucleotide sequence comprising SNP rs10490924; (c) a CFHnucleotide sequence comprising SNP rs800292; or (d) a complementcomponent 2 (C2) nucleotide sequence comprising SNP rs547154.These SNPs are risk factors for ocular conditions.In an example, the ocular condition is DR or ROP and the method is fortreating or reducing the risk of DR or ROP.11. The method of any one of concepts 2 to 6, wherein the humancomprises (i) an ARMS2 nucleotide sequence comprising a G at theposition of SNP rs10490924; (ii) a CFH nucleotide sequence comprising aT at the position of SNP rs1061170 or rs3766404; (iii) a prothrombinnucleotide sequence comprising SNP rs1799963; (iv) a MTHFR nucleotidesequence comprising SNP rs1801133; (v) a Factor V nucleotide sequencecomprising SNP rs6025; or (vi) a VEGFR2 nucleotide sequence comprisingSNP rs4576072 or rs6828477.These SNPs are associated with improved responses following treatment(eg, PDT and/or VEGF-A antagonism) of an ocular disease or condition.In an example, the ocular condition is DR and the method is for treatingor reducing the risk of DR.

SNPs Associated With Improved Responses Following Anti-VEGF-A TreatmentOf An Ocular Disease Or Condition

12. A method of treating or reducing the risk of a disease or conditionmediated by VEGF-A in a human, the method comprising administering tosaid human an anti-VEGF-A ligand (eg, an anti-VEGF-A trap, antibody orantibody fragment) that specifically binds to a human VEGF-A, whereinthe human comprises (i) an ARMS2 nucleotide sequence comprising a G atthe position of SNP rs10490924; (ii) a CFH nucleotide sequencecomprising a T at the position of SNP rs1061170 or an T at the positionof rs3766404; or (iii) a VEGFR2 nucleotide sequence comprising SNPrs4576072 or rs6828477, wherein the ligand comprises a human gamma-1heavy chain constant region that comprises an amino acid selected fromthe group consisting of an Asp corresponding to position 204 of SEQ IDNO: 42 and a Leu corresponding to position 206 of SEQ ID NO: 42 andwherein said human comprises an IGHG1*01 human heavy chain constantregion gene segment, or the human expresses antibodies comprising humangamma-1 heavy chain constant regions comprising said selected aminoacid.These SNPs are associated with improved responses following anti-VEGF-Atreatment of an ocular disease or condition.In an example, the ocular condition is DR and the method is for treatingor reducing the risk of DR.In an example the human comprises an ARMS2 nucleotide sequencecomprising G at the position of SNP rs10490924, eg, in homozygous form.In an example the human comprises a CFH nucleotide sequence comprising aT at the position of SNP rs1061170, eg, in homozygous form.In an example the human comprises a CFH nucleotide sequence comprising aT at the position of SNP rs3766404, eg, in homozygous form.In an example the human comprises a VEGFR2 nucleotide sequencecomprising SNP rs4576072, eg, in homozygous form.In an example the human comprises a VEGFR2 nucleotide sequencecomprising SNP rs6828477, eg, in homozygous form.

-   13. The method of concept 12, wherein the condition is an ocular    condition, eg, diabetic retinopathy or retinopathy of prematurity.    In an example, the condition is diabetic retinopathy (DR).    In an example, the condition is age-related macular degeneration    (AMD), eg, exudative AMD.    In an example, the condition is polypoidal choroidal vasculopathy    (PCV).    In an example, the condition is retinal angiomatous proliferation    (RAP).    In an example, the condition is diabetic macular edema (DME).    In an example, the condition is retinal-vein occlusion (RVO).    In an example, the condition is retinopathy of prematurity (ROP).    In an alternative, instead of “a method of treating or reducing the    risk of a disease or condition mediated by VEGF-A in a human”, the    invention relates to “a method of improving visual acuity in a    human”.    14. The method of concept 12 or 13, wherein the human further    comprises a VEGF-A SNP selected from the group consisting of    rs699947, rs833061, rs2010963, rs3025039 and rs699946.    In an example, the SNP is rs699947.    In an example, the SNP is rs833061.    In an example, the SNP is rs2010963.    In an example, the SNP is rs3025039.    In an example, the SNP is rs699946.    15. The method of any one of concepts 12 to 14, wherein the method    further comprises treating the human with photodynamic therapy.

For example, the method further comprises treating the human with PDTsimultaneously with, or after, administration of said ligand to thehuman.

16. The method of concept 12, wherein the disease or condition is acancer.

17. The method of concept 16, wherein the human further comprises VEGF-ASNP rs699947 or rs1570360.

In an example, the SNP is rs699947, eg, homozygous for said SNP.

In an example, the SNP is rs1570360, eg, homozygous for said SNP.

Antagonism of VEGF-A & PDGF-B Pathways

PDGF-B is also known as IBGC5, PDGF-2, PDGF2, SIS, SSV and c-sis.Platelet-derived growth factor receptor beta (PDGFRB) is also known asCD140B, IBGC4, IMF1, JTK12, PDGFR-1 and PDGFR1.

PDGF is a growth factor which may play a role in ocularneovascularization, and has been documented to be a mitogen forpericytes, as well as smooth muscles cells, fibroblasts, and othermesenchymal cell types. Pericytes appear critical for the maintenance ofestablished blood vessels and may be important in the maturation processin ocular conditions, such as choroidal neovascularization. It has beenhypothesized that there is a period of VEGF dependency of endothelialcells that overlaps with a period of responsiveness to PDGF withdrawalin newly formed neovascular vessels. Established mature vessels may beassociated with resistance to anti-VEGF blockade or rapid recurrence ofchoroidal neovascularization.

PDGF has been demonstrated to stimulate angiogenesis and pericyterecruitment. Loss of pericytes in retinal vessels is thought to beassociated with abnormalities of the vasculature and instability,including the formation of microaneurysms and vascular permeability.Loss of pericytes with PDGF-blockade may be associated with regressionof maturing neovascularization. Pericytes produce VEGF-A under selectedconditions, possibly thereby protecting endothelial cells whengeneralized suppression of VEGF-A is present.

Reference is made to Am J Pathol. 2006 June; 168(6):2036-53; “Inhibitionof platelet-derived growth factor B signaling enhances the efficacy ofanti-vascular endothelial growth factor therapy in multiple models ofocular neovascularisation”; Jo N et al. Jo et al demonstrated increasedsuppression of neovascularization in experimental models with specificblockade of both VEGF-A and PDGF-B as compared with blockade with VEGF-Aalone. In addition, neovascularization became more resistant to VEGF-Ablockade with progression. Suppression of VEGF-A and PDGF-B togethercaused regression of neovascularization not responsive to anti-VEGF-Aalone.

Reference is also made to:—

-   -   Angiogenesis. 2014 July; 17(3):553-62. doi:        10.10071s10456-013-9402-5. Epub 2013 Oct. 24;    -   “Antagonism of PDGF-BB suppresses subretinal neovascularization        and”; enhances the effects of blocking VEGF-A2; Dong A et al    -   MAbs. 2010 January-February.; 2(1):20-34. Epub 2010 Jan. 2; “A        dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable        antibody fragments demonstrates anti-angiogenic activity in        vitro and in vivo”; Mabry R et al.

A treatment regimen combining inhibition of pericyte and endothelialcell function through the use of concomitant administration ofanti-VEGF-A targeting and PDGF-BB pathway targeting is useful forblocking both paracrine and autocrine VEGF signalling in endothelialcells.

Thus, this aspect of the invention provides the following.

18. The method of any preceding concept, wherein the method furthercomprises antagonising PDGF-B in said human.

In an example of any aspect herein, the method comprises administeringREGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human. REGN2176-3comprises aflibercept.

19. The method of concept 18, wherein the method comprises administeringan anti-PDGF-B ligand to the human simultaneously or sequentially withthe anti-VEGF-A ligand.

In an example, the anti-VEGF-A and anti-PDGF-B ligands are comprisedtogether by a pharmaceutical formulation that is administered to thehuman. In an example of any aspect herein, the method comprisesadministering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the human.In an example a bispecific ligand (eg, trap or antibody or antibodyfragment) is administered, wherein the ligand comprises a human VEGF-Abinding site and a huan PDGF-B binding site.In an example, the anti-PDGF-B ligand antagonises a human PDGF-B encodedby said PDGF-B nucleotide sequence of (i).In an example, the anti-PDGF-B ligand comprises or consists of ananti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an anti-PDGF-Baptamer (eg, E10030 or FOVISTA™) or an anti-PDGF-B NCE (so-called NewChemical Entity in the art).Examples of suitable anti-PDGF-B pathway antibodies and variable regionsfor incorporation into antibodies for use in the invention are disclosedin W02014109999A1, the disclosure of which, and specifically includingthe sequences therein of such antibodies and variable regions, as wellas methods of testing and medical diseases and conditions for use (whichare suitable for the present invention), are incorporated herein asthough explicitly written herein and to provide disclosure of featuresthat can be incorporated into concepts herein.In an example herein, the anti-PDGF-B ligand specifically binds to humanPDGF-B dimer (ie, so-called PDGF-BB).20. The method of concept 18, wherein the method comprises administeringan anti-PDGFR-B ligand to the human simultaneously or sequentially withthe anti-VEGF-A ligand.In an example, the anti-VEGF-A and anti-PDGFR-B ligands are comprisedtogether by a pharmaceutical formulation that is administered to thehuman. In an example a bispecific ligand (eg, trap or antibody orantibody fragment) is administered, wherein the ligand comprises a humanVEGF-A binding site and a human PDGFR-B binding site. Reference is madeto Dong et al, which describes a suitable example.In an example, the PDGFR-B ligand antagonises a human PDGFR-B encoded bysaid PDGF-B nucleotide sequence of (ii).In an example herein, the anti-PDGFRB antagonises binding of thereceptor to human PDGF-B dimer (ie, so-called PDGF-BB), eg, wherein thePDGF-B is encoded by a nucleotide sequence of (i).Suitable PDGFRβ-specific inhibitors are disclosed in WO2008130704, forexample, the disclosure and sequences of which are incorporated hereinby reference.21. The method of any preceding concept, wherein the human has receiveda human PDGF-B or PDGFR-B antagonist prior to administering saidanti-VEGF-A ligand.

Antagonism of VEGF-A & Angiopoietin-2 Pathways

The angiopoietins are ligands for the endothelial cell receptor Tie2 andare essential for vascular development and angiogenesis. Unlike otherfamily members, angiopoietin-2 (Ang2) is strongly up-regulated byendothelial cells at sites of angiogenesis and vascular remodelling,including tumours Enhanced anti-tumour effects have been observed inpreclinical models with combined blockade of both VEGF and Ang2.

The Ang2 pathway acts to remodel blood vessels and, in particular, isactive during both developmental and pathological angiogenesis. But, itis nearly absent from mature, quiescent vasculature. Hence a combinationAng2/VEGF blockade results in decreased survival and maturation ofneovessels as well as inhibition of pathological leaks. Ang2 inhibitionalso blocks vascular remodelling driven by inflammation in model systemsin which VEGF inhibition is ineffective, suggesting that combinationblockade has potential for a greater therapeutic benefit.

The invention thus further provides:—

22. The method of any one of concepts 1 to 17, comprising antagonisingangiopoietin-2 (Ang2) in said human.

In an example, the method comprises administering nesvacumab (RegeneronPharmaceuticals, Inc) to the human.

23. The method of concept 22, wherein the method comprises administeringan anti-Ang2 ligand (eg, nesvacumab) to the human simultaneously orsequentially with the anti-VEGF-A ligand.

In an example, the anti-Ang2 ligand comprises or consists of ananti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2 aptameror an anti-Ang2 NCE (so-called New Chemical Entity in the art).

24. The method of concept 22, wherein an anti-Ang2 receptor (eg,anti-human Tie2) ligand is administered to the human simultaneously orsequentially with the anti-VEGF-A ligand.

25. The method of any one of concepts 1 to 24, wherein the human hasreceived a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior toadministering said anti-VEGF-A ligand.

26. The method of any preceding concept, wherein the anti-VEGF-A ligand(eg, an anti-VEGF-A antibody, antibody fragment or VEGF-A trap)comprises a human gamma-1 heavy chain constant region that comprises anamino acid selected from the group consisting of an Asp corresponding toposition 204 of SEQ ID NO: 42 and a Leu corresponding to position 206 ofSEQ ID NO: 42; and wherein said human comprises an IGHG1*01 human heavychain constant region gene segment, or the human expresses antibodiescomprising human gamma-1 heavy chain constant regions comprising saidselected amino acid.Optionally, this constant region is comprised by an Fc region of theligand.In one embodiment, the ligand of the invention is a VEGF-A trapcomprising the polypeptide of SEQ ID NO: 127, eg, comprising orconsisting of the polypeptide of SEQ ID NO: 127.27. The method of any one of concepts 1 to 25, wherein the anti-VEGF-Aligand (eg, an anti-VEGF-A antibody, antibody fragment or VEGF-A trap)comprises a human gamma-2 heavy chain constant region that comprises anamino acid selected from the group consisting of a Pro corresponding toposition 72 of SEQ ID NO: 44, an Asn corresponding to position 75 of SEQID NO: 44, a Phe corresponding to position 76 of SEQ ID NO: 44, a Valcorresponding to position 161 of SEQ ID NO: 44 and an Ala correspondingto position 257 of SEQ ID NO: 44 and wherein said human comprises anIGHG2*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-2 heavy chain constantregions comprising said selected amino acid.Optionally, this constant region is comprised by an Fc region of theligand.For example, the ligand is a VEGF-A trap comprising one or more humanVEGF-A receptor domain sequences fused to a human gamma-1 or -2 heavychain constant region, eg, a constant region as defined in concept 26 or27. In an example, such a trap comprises first and second (eg, comprisesonly two species of) VEGF-A receptor domain sequences, eg, first andsecond human Flt1 domain sequences; or first and second human KDR domainsequences; or a Flt domain sequence and a KDR domain sequence. In anexample, the trap comprises a dimer of the following polypeptide (N- toC-terminally): a human Flt1 Ig-like C2-type 2 domain sequence and ahuman KDR Ig-like C2-type 3 domain sequence fused to the human gamma-1heavy chain constant region of concept 26 or human gamma-2 heavy chainconstant region of concept 27. In an example, the constant regions arepart of antibody Fc regions.A suitable human Flt1 Ig-like C2-type 2 domain sequence for use in thepresent invention is shown in SEQ ID NO: 129. A suitable human KDRIg-like C2-type 3 domain sequence for use in the present invention isshown in SEQ ID NO: 130.28. The method of any one of concepts 1 to 27, wherein the anti-VEGF-Aligand is a human anti-VEGF-A antibody, antibody fragment or VEGF-Atrap.29. The method of any preceding concept, comprising, before saidadministering, selecting a human comprising said VEGF-A, HTRA1, ARMS2,CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP, wherein the humanis the human of concept 1 or 12.30. The method of any one of concepts 1 to 28, wherein the human hasbeen determined to comprise said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR,prothrombin, factor V or VEGFR2 SNP.31. The method of any one of concepts 1 to 28, comprising the step ofdetermining that the human comprises said VEGF-A, HTRA1, ARMS2, CFH, C2,MTHFR, prothrombin, factor V or VEGFR2 SNP, optionally, wherein thedetermining step is performed before administration of the ligand to thehuman.32. The method of concept 31, wherein the step of determining comprisesassaying a biological sample from the human for said VEGF-A, HTRA1,ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP.33. The method of concept 32, wherein the assaying comprises nucleicacid amplification and optionally one or more methods selected fromsequencing, next generation sequencing, nucleic acid hybridization, andallele-specific amplification and/or wherein the assaying is performedin a multiplex format.34. An anti-VEGF-A ligand (eg, an anti-VEGF-A antibody, antibodyfragment or VEGF-A trap) for use in a method of any one of concepts 1 to33.35. The ligand of concept 34, wherein the anti-VEGF-A ligand is a humananti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg,aflibercept).36. The ligand of concept 34 or 35, wherein said ligand is administeredby intravenous or subcutaneous administration and/or is comprised in aninjectable preparation.

C2 SNP rs547154 is indicated in the 1000 Genomes database as having anaverage cumulative allele frequency of 10% across all human populations;a frequency for the AFR population of 15% and above average also for thefollowing human populations: PUR, IBS, GBR, ASW, LWK, YRI. Thus, whenthe ancestry of the human belongs to one of these populations, the humanmay have an elevated risk of an ocular disease or condition or otherVEGF-A mediated disease or condition and the invention comprisesadministering the ligand to such a human. Thus, in an embodiment, thehuman comprises C2 SNP rs547154 and is of AFR, PUR, IBS, GBR, ASW, LWKor YRI ancestry. For example, the human is of AFR ancestry.

VEGF-A SNP rs1413711 is indicated in the 1000 Genomes database as havingan average cumulative allele frequency of 64% across all humanpopulations; a frequency for the AFR and ASN populations of 80% and 71%respectively, and above average also for the following humanpopulations: ASW, LWK, YRI, PUR, CHB, CHS and JPT. Thus, when theancestry of the human belongs to one of these populations, the human mayhave an improved treatment of an ocular disease or condition and theinvention comprises administering the ligand to such a human. Thus, inan embodiment, the human comprises VEGF-A SNP rs1413711 (eg, inhomozygous form) and is of AFR, ASN, ASW, LWK, YRI, PUR, CHB, CHS or JPTancestry. For example, the human is of AFR ancestry. For example, thehuman is of ASN ancestry. In an example, the method is for improvingvisual acuity in said human.

VEGF-A SNP rs699946 is indicated in the 1000 Genomes database as havingan average cumulative allele frequency of 26% across all humanpopulations; a frequency for the ASN population of 42%, and aboveaverage also for the following human populations: PUR 34%, CHB 45%, CHS41% and JPT 40%. Thus, when the ancestry of the human belongs to one ofthese populations, the human may have an improved treatment of an oculardisease or condition and the invention comprises administering theligand to such a human. Thus, in an embodiment, the human comprisesVEGF-A SNP rs699946 (eg, in homozygous form) and is of ASN, ASW, PUR,CHB, CHS or JPT ancestry. For example, the human is of ASN ancestry. Inan example, the method is for improving visual acuity in said human.

VEGF-A SNP rs3025000 is indicated in the 1000 Genomes database as havingan average cumulative allele frequency of 26% across all humanpopulations; a frequency for the AMR, ASN and EUR populations of 30%,42% and 27% respectively, and above average also for the following humanpopulations: MXL (29%), PUR (38%), CHB (45%), CHS (41%, JPT (40%), CEU(30%), GBR (27%), IBS (39%) and TSI (31%). Thus, when the ancestry ofthe human belongs to one of these populations, the human may have animproved treatment of an ocular disease or condition and the inventioncomprises administering the ligand to such a human. Thus, in anembodiment, the human comprises VEGF-A SNP rs3025000 (eg, in homozygousform) and is of AMR, ASN, EUR, MXL, PUR, CHB, CHS, JPT, CEU, GBR, IBS orTSI ancestry. For example, the human is of AMR ancestry. For example,the human is of ASN ancestry. For example, the human is of EUR ancestry.

Prothrombin SNP rs1799963 is almost exclusively in Caucasians; this SNPis indicated in the 1000 Genomes database as having an above averagecumulative allele frequency in the following human populations: AMR,EUR, TSI, GBR and PUR. Thus, when the ancestry of the human belongs toone of these populations, the human may have an improved treatment of anocular disease or condition and the invention comprises administeringthe ligand to such a human. Thus, in an embodiment, the human comprisesprothrombin SNP rs1799963 (eg, in homozygous form) and is of Caucasian,AMR, EUR, TSI, GBR or PUR ancestry. For example, the human is ofCaucasian ancestry. For example, the human is of EUR ancestry. In anexample, the condition is CNV.

MTHFR SNP rs1801133 is indicated in the 1000 Genomes database as havingan above average cumulative allele frequency in the following humanpopulations: AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PUR and TSI. Thus,when the ancestry of the human belongs to one of these populations, thehuman may have an improved treatment of an ocular disease or conditionand the invention comprises administering the ligand to such a human.Thus, in an embodiment, the human comprises MTHFR SNP rs1801133 (eg, inhomozygous form) and is of AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PURor TSI ancestry. For example, the human is of Caucasian ancestry. Forexample, the human is of AMR ancestry. For example, the human is of ASNancestry. For example, the human is of EUR ancestry. For example, thehuman is of EUR ancestry. In an example, the condition is CNV.

VEGFR2 SNP rs4576072 is indicated in the 1000 Genomes database as havingan average cumulative allele frequency of 8% across all humanpopulations; a frequency for the AMR and EUR populations of 10% and 11%respectively, and above average also for the following humanpopulations: PUR (14%), CEU (9%), GBR (13%) and TSI (13%). Thus, whenthe ancestry of the human belongs to one of these populations, the humanmay have an improved treatment of an ocular disease or condition and theinvention comprises administering the ligand to such a human. Thus, inan embodiment, the human comprises VEGFR2 SNP rs4576072 (eg, inhomozygous form) and is of AMR, EUR, PUR, CEU, GBR or TSI ancestry. Forexample, the human is of AMR ancestry. For example, the human is of EURancestry. In an embodiment 3 or 4 VEGFR2 SNPs from rs4576072 andrs6828477 provide for improved treatment. Thus, for example, the humancomprises 3 or 4 copies of VEGFR2 SNPs selected from rs4576072 andrs6828477, eg, the human comprise GG (ie, the human comprises VEGFR2 SNPrs4576072 in homozygous form) and AA or AG (ie, the human comprisesVEGFR2 SNP rs6828477, eg, in homozygous form); or GA (rs4576072) andAA(rs6828477).

Administering Human VEGF-A Ligands to Humans Comprising PDFG-B PathwaySNP Risk Factors

Endothelial cells (ECs) in blood vessels under formation are stabilizedby the recruitment of pericytes, both in normal tissues and duringangiogenesis in pathologic situations, including cancer. Endothelialcells in small blood vessels and capillaries interact with pericytes,cells of mesenchymal origin that provide important support for bloodvessel formation and function. Numerous studies describe thesignificance of members of the platelet-derived growth factor (PDGF)family for recruitment to and productive association of pericytes withthe blood vessel endothelium during embryonal development. Strikingly,mice lacking components of the PDGF-B/PDGFR-β signaling axis areessentially devoid of pericytes, and as a consequence die frommicrohemorrhaging because of malformed blood vessels. Reference is madeto Blood. 2011 Sep. 8; 118(10): 2906-2917, prepublished online 2011 July21, doi: 10.1182/blood-2011-01-331694, “Pericytes promote endothelialcell survival through induction of autocrine VEGF-A signaling and Bcl-wexpression”, Franco M et al. The authors describe that newly formedvascular sprouts recruit pericytes primarily through secretion ofplatelet derived growth factor beta (PDGF-BB) by endothelial tip cells.Pericytes respond by migrating along the matrix-retained gradient ofPDGF-BB, and by a proliferative burst as they reach the tip of thesprout. Reciprocally, pericytes up-regulate expression of cell-boundVEGF-A as they mature and are recruited into newly formed vessels. Theauthors found that pericytes, in addition to producing VEGF-A that actsin a paracrine fashion, stimulate autocrine expression of VEGF-A bytumour endothelial cells. The authors suggest that a treatment regimencombining inhibition of pericyte and endothelial cell function throughthe use of concomitant administration of a VEGF-targeting antibody and aPDGF-BB/PDGFRP-targeting antibody for blocking both paracrine andautocrine VEGF signalling in ECs.

The invention thus provides the following aspects:—

-   1. A method of treating or reducing the risk of a VEGF-A mediated    disease or condition in a human, the method comprising administering    an anti-human VEGF-A ligand; an anti-human PDGF-B ligand or an    anti-PDGFR-B ligand to a human, wherein the human comprises (i) a    PDGF-B nucleotide sequence comprising a SNP selected from the group    consisting of rs142404523 (ie, a C corresponding to position −776)    and a C at the position of rs1800818 (ie, a C corresponding to    position −735); or (ii) a PDGFR-B nucleotide sequence comprising a    SNP selected from the group consisting of rs246395 (ie, a G    corresponding to position 2601) and rs74943037 (ie, a T    corresponding to position 1391).    -   A suitable example of an anti-VEGF-A ligand, which is an        antagonist of human VEGF-A, is aflibercept, sunitinib, ESBA1008,        bevacizumab or ranizumab. In an example, the ligand comprises or        consists of an anti-VEGF-A antibody or fragment (eg, ESBA1008,        bevacizumab or ranizumab), an anti-VEGF-A trap (eg, VEGF-A        receptor binding sites fused to antibody Fc regions, eg,        aflibercept), an aptamer or an NCE (so-called New Chemical        Entity in the art, eg, sunitinib).    -   In an example of any aspect herein, the method comprises        administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the        human. REGN2176-3 comprises aflibercept and an anti-PDGF-B        ligand.    -   In an embodiment, said selected SNP (eg, rs246395) is associated        with VEGF-A secretion from pericytes in said human, for example        maintained or increased VEGF-A secretion from pericytes in said        human (eg, ocular pericytes or pericytes at the site of a solid        tumour in said patient). Thus, administration of an anti-VEGF-A        ligand would be beneficial to also target such secreted VEGF-A.        This is useful, for example, when the disease or condition is        angiogenesis or neovascularisation or associated with one or        both of these.    -   For PDGF-B, a C at the position of rs1800818 (ie, −735C) is        indicated in the 1000 Genomes database as having an average        cumulative allele frequency of 37% across all human populations;        a frequency for the AFR and EUR populations of 62% and 41%        respectively (ie, above average) and also above average for the        following human populations: ASW 60%, LWK 58%, YRI 68%, CEU 46%,        FIN 46%, IBS 50% and TSI 38%. Thus, when the ancestry of the        human belongs to one of these populations, the human may have an        elevated risk of angiogenesis or other VEGF-A mediated disease        or condition and the invention comprises administering the        ligand such a human. Thus, in an embodiment, the human comprises        a PDGF-B nucleotide sequence comprising −735C, ie, a C at the        position of PDGF-B SNP rs1800818 and wherein the human is of        AFR, EUR, ASW, LWK, YRI, CEU, FIN, IBS or TSI ancestry. For        example, the human is of AFR ancestry. For example, the human is        of EUR ancestry.    -   For PDGF-B, SNP rs142404523 (−776T>C) is indicated in the 1000        Genomes database as having an average cumulative allele        frequency of 8% across all human populations; a frequency for        the AFR and EUR populations of 17% and 10% respectively (ie,        above average) and also above average for the following human        populations: ASW 16%, LWK 18%, YRI 16%, PUR 11%, CEU 11%, FIN        9%, GBR 12% and IBS 18%. Thus, when the ancestry of the human        belongs to one of these populations, the human may have an        elevated risk of angiogenesis or other VEGF-A mediated disease        or condition and the invention comprises administering the        ligand such a human.    -   Thus, in an embodiment, the human comprises PDGF-B SNP        rs142404523 and is of AFR, EUR, ASW, LWK, YRI, PUR, CEU, FIN,        GBR or IBS ancestry. For example, the human is of AFR ancestry.        For example, the human is of EUR ancestry.    -   For PDGFR-B, SNP rs246395 (2601A>G) the 1000 Genomes database        indicates an average cumulative allele frequency of 23% for G        across all human populations; a frequency for the AMR and EUR        populations of 29% and 30% respectively (ie, above average) and        also above average for the following human populations: CLM        (36%), MXL (25%), PUR (26%), CEU (39%), GBR (30%), IBS (29%) and        TSI (31%). Thus, when the human belongs to one of these        populations, it may have an elevated risk of angiogenesis or        other VEGF-A mediated disease or condition and the invention        comprises administering the ligand such a human. Thus, in an        embodiment, the human comprises PDGFR-B SNP rs246395 and is of        AMR, EUR, CLM, MXL, PUR, CEU, GBR, IBS or TSI ancestry. For        example, the human is of AMR ancestry. For example, the human is        of EUR ancestry.    -   For PDGFR-B, SNP rs74943037 is indicated in the 1000 Genomes        database as having an average cumulative allele frequency of 3%        for G across all human populations; a frequency for the AFR        population of 10% (ie, above average) and also above average for        the following human populations: ASW 8%, LWK 14% and YRI 8%.        Thus, when the ancestry of the human belongs to one of these        populations, the human may have an elevated risk of angiogenesis        or other VEGF-A mediated disease or condition and the invention        comprises administering the ligand such a human. Thus, in an        embodiment, the human comprises PDGFR-B SNP rs74943037 and is of        AFR, ASW, LWK or YRI ancestry. For example, the human is of AFR        ancestry.-   2. The method of aspect 1, wherein the human is homozygous for said    selected SNP (eg, homozygous for rs246395 or homozygous for a C at    the position of rs1800818 (ie, homozygous for a C corresponding to    position −735)).    Such homozygotes may have increased severity or risk of said disease    or condition.-   3. The method of aspect 1 or 2, wherein the human comprises a PDGF-B    nucleotide sequence comprising a SNP of (i) and a PDFGR-B nucleotide    sequence comprising a SNP of (ii).    For example, the human comprises a C at the position of rs1800818    and rs246395. In another example, the human comprises a C at the    position of rs1800818 and rs74943037.    For example, the human comprises rs142404523 and rs246395. In    another example, the human comprises rs142404523 and rs74943037.-   4. The method of any one of aspects 1 to 3, wherein the human    comprises (a) a VEGF-A nucleotide sequence comprising a SNP selected    from the group consisting of rs699947, rs833061, rs2010963,    rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069,    rs3025000 and rs1570360; (b) a HTRA1 nucleotide sequence comprising    SNP rs11200638; (c) an ARMS2 nucleotide sequence comprising SNP    rs10490924; (d) a Complement Factor H (CFH) nucleotide sequence    comprising SNP rs800292, rs1061170 or rs3766404; (e) a Complement    Component 2 (C2) nucleotide sequence comprising SNP rs547154; (0 a    prothrombin nucleotide sequence comprising SNP rs1799963; (g) a    MTHFR nucleotide sequence comprising SNP rs1801133; (h) a Factor V    nucleotide sequence comprising SNP rs6025; or (i′) a VEGFR2    nucleotide sequence comprising SNP rs4576072 or rs6828477.    In an example, the human comprises a SNP of (a).    For example, the human comprises rs699947 and optionally the    condition is DR or a solid tumour.    For example, the human comprises rs833061 and optionally the    condition is DR or a solid tumour.    For example, the human comprises rs2010963 and optionally the    condition is DR or a solid tumour.    For example, the human comprises rs3025039 and optionally the    condition is DR or a solid tumour.    For example, the human comprises rs699946 and optionally the    condition is DR or a solid tumour.    For example, the human comprises rs2146323.    For example, the human comprises rs1413711, optionally the human is    homozygous for said SNP.    For example, the human comprises rs833068, optionally the human is    homozygous for said SNP.    For example, the human comprises rs833069, optionally the human is    homozygous for said SNP.    For example, the human comprises rs3025000.    For example, the human comprises rs1570360.    In an example, the human comprises a SNP of (a) and (b). In an    example, the human comprises a SNP of (a) and (c). In an example,    the human comprises a SNP of (a) and (d). In an example, the human    comprises a SNP of (a) and (e). In an example, the human comprises a    SNP of (a) and (f). In an example, the human comprises a SNP of (a)    and (g). In an example, the human comprises a SNP of (a) and (h). In    an example, the human comprises a SNP of (a) and (i′).    Optionally in any aspect herein, the ligand specifically binds to or    antagonises a VEGF-A, eg, a VEGF-A encoded by a VEGF-A nucleotide    sequence comprising a SNP of (a). In one example, this can be    determined by specific binding to or antagonism of VEGF-A in a    sample in vitro (eg, serum or blood sample) from the human, wherein    the human comprises said VEGF-A nucleotide sequence. Specific    binding can be determined, for example, by SPR or ELISA as will be    known to the skilled person. The skilled person will also be    familiar with standard assays for VEGF-A antagonism. Additionally,    in an example the genome of the human comprises said SNP of (a).-   5. The method of any one of aspects 1 or 4, wherein the human    comprises a human VEGF-A nucleotide sequence comprising a SNP    selected from the group consisting of rs699947, rs833061, rs2010963,    rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069,    rs3025000 and rs1570360 and the ligand antagonises a VEGF-A encoded    by said nucleotide sequence.    In an example of any aspect herein, the VEGF-A is VEGF-A-(165)    isoform.-   6. The method of any one of aspects 1 to 5, wherein the method    further comprises antagonising PDGF-B in said human.    In an example of any aspect herein, the method comprises    administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the    human. REGN2176-3 comprises aflibercept.-   7. The method of aspect 6, wherein the method comprises    administering an anti-PDGF-B ligand to the human simultaneously or    sequentially with the anti-VEGF-A ligand.    In an example, the anti-VEGF-A and anti-PDGF-B ligands are comprised    together by a pharmaceutical formulation that is administered to the    human. In an example of any aspect herein, the method comprises    administering REGN2176-3 (Regeneron Pharmaceuticals, Inc) to the    human.    In an example, the anti-PDGF-B ligand antagonises a human PDGF-B    encoded by said PDGF-B nucleotide sequence of (i).    In an example, the anti-PDGF-B ligand comprises or consists of an    anti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an    anti-PDGF-B aptamer (eg, E10030 (FOVISTA™), described in, e.g., U.S.    Pat. Nos. 6,207,816; 5,731,144; 5,731,424; and 6,124,449; each of    which is incorporated. by reference herein in its entirety) or an    anti-PDGF-B NCE (so-called New Chemical Entity in the art).    Examples of suitable anti-PDGF-B pathway antibodies and variable    regions for incorporation into antibodies for use in the invention    are disclosed in WO2014109999A1, the disclosure of which, and    specifically including the sequences therein of such antibodies and    variable regions, as well as methods of testing and medical diseases    and conditions for use (which are suitable for the present    invention), are incorporated herein as though explicitly written    herein and to provide disclosure of features that can be    incorporated into aspects herein.    In an example herein, the anti-PDGF-B ligand specifically binds to    human PDGF-B dimer (ie, so-called PDGF-BB).-   8. The method of aspect 6, wherein the method comprises    administering an anti-PDGFR-B ligand to the human simultaneously or    sequentially with the anti-VEGF-A ligand.    In an example, the anti-VEGF-A and anti-PDGFR-B ligands are    comprised together by a pharmaceutical formulation that is    administered to the human.    In an example, the PDGFR-B ligand antagonises a human PDGFR-B    encoded by said PDGF-B nucleotide sequence of (ii).    In an example herein, the anti-PDGFRB antagonises binding of the    receptor to human PDGF-B dimer (ie, so-called PDGF-BB), eg, wherein    the PDGF-B is encoded by a nucleotide sequence of (i).    Suitable PDGFRP-specific inhibitors are disclosed in WO2008130704,    for example, the disclosure and sequences of which are incorporated    herein by reference.-   9. The method of any one of aspects 1 to 8, wherein the human has    received a human PDGF-B or PDGFR-B antagonist prior to administering    said anti-VEGF-A ligand.-   10. The method of any one of aspects 1 to 9, wherein the condition    is selected from the group consisting of angiogenesis,    neovascularisation, ocular vascularisation and solid tumour    vascularisation.    In an example, the condition is subretinal neovascularisation.-   11. The method of any one of aspects 1 to 9, wherein the disease or    condition is an angiogenic disease or condition.    Optionally the disease or condition is an angiogenic ocular    condition or angiogenic cancer, eg a solid tumour, a    gastrointestinal cancer, colorectal cancer, liver cancer, breast    cancer, ovarian cancer, non small cell lung cancer, thyroid cancer,    an oral cancer, or an haematological cancer.    Optionally the angiogenic disease or condition is selected from the    group consisting of psoriasis, rheumatoid arthritis, a hemangioma,    an angiofibroma, diabetic retinopathy, corneal neovacularisation and    neovascular glaucoma.-   12. The method of any one of aspects 1 to 11, wherein the disease or    condition is or comprises vascular permeability (eg, ocular vascular    permeability), edema or inflammation in said human.    For example, the disease or condition is selected from the group    consisting of brain edema (eg, edema associated with brain injury,    stroke or brain tumour); edema associated with an inflammatory    disorder (eg, associated with psoriasis or arthritis, rheumatoid    arthritis or asthma); edema associated with burns; ascites or    pleural effusion (eg, associated with tumours, inflammation or    trauma); chronic airway inflammation; capillary leak syndrome;    sepsis; kidney disease (eg, associated with increased leakage of    protein); and an eye disorder (eg, age related macular degeneration    or diabetic retinopathy).-   13. The method of any one of aspects 1 to 12 for reducing or    reducing the risk of VEGF-A mediated endothelial cell maintenance or    proliferation in an angiogenic disease or condition in said human.    In an example, the angiogenic disease or condition is an angiogenic    disease or condition recited above, eg, an angiogenic ocular    condition.-   14. The method of any one of aspects 1 to 12 for regressing    neovascularisation in a VEGF-A mediated disease or condition in said    human.-   15. The method of any one of aspects 1 to 12 for treating or    reducing the risk of neovascularisation in a human, wherein the    human has VEGF-A ligand-refractory neovascularisation.    For example, the human is partially or completely resistant to    treatment with an anti-VEGF-A ligand, eg, aflibercept, sunitinib,    bevacizumab or ranizumab.-   16. The method of any one of aspects 1 to 15, comprising    antagonising angiopoietin-2 (Ang2) in said human.    The angiopoietins are ligands for the endothelial cell receptor Tie2    and are essential for vascular development and angiogenesis. Unlike    other family members, angiopoietin-2 (Ang2) is strongly up-regulated    by endothelial cells at sites of angiogenesis and vascular    remodelling, including tumours. Enhanced anti-tumour effects have    been observed in preclinical models with combined blockade of both    VEGF and Ang2.    In an example, the method comprises administering nesvacumab    (Regeneron Pharmaceuticals, Inc) to the human.-   17. The method of aspect 16, wherein the method comprises    administering an anti-Ang2 ligand (eg, nesvacumab) to the human    simultaneously or sequentially with the anti-VEGF-A ligand.    In an example, the anti-Ang2 ligand comprises or consists of an    anti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2    aptamer or an anti-Ang2 NCE (so-called New Chemical Entity in the    art).-   18. The method of aspect 16, wherein an anti-Ang2 receptor (eg,    anti-human Tie2) ligand is administered to the human simultaneously    or sequentially with the anti-VEGF-A ligand.-   19. The method of any one of aspects 1 to 18, wherein the human has    received a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior    to administering said anti-VEGF-A ligand.-   20. An anti-TOI (eg, VEGF-A) ligand (eg, an antibody, antibody    fragment or TOI trap) for use in a method of any one of aspects 1 to    19.-   21. The ligand of aspect 20, wherein the ligand is a human    anti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg,    aflibercept).-   22. The ligand of aspect 20 or 21, wherein said ligand is for    intravenous or subcutaneous administration and/or is comprised in an    injectable preparation.

General Optional Features Applicable to all Examples, Embodiments,Concepts & Aspects

There is optionally provided the method or ligand wherein the anti-TOI(eg, VEGF-A) ligand (eg, an antibody, antibody fragment or TOI trap)comprises a human gamma-1 heavy chain constant region that comprises anAsp corresponding to position 204 of SEQ ID NO: 42 or a Leucorresponding to position 206 of SEQ ID NO: 42 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp corresponding to position204 of SEQ ID NO: 42 or a Leu corresponding to position 206 of SEQ IDNO: 42.

For example, the ligand is a VEGF-A trap comprising one or more humanVEGF-A receptor domain sequences fused to a human gamma-1 heavy chainconstant region, eg, a constant region as defined in the immediatelypreceding paragraph. In an example, such a trap comprises first andsecond (eg, comprises only two) VEGF-A receptor domain sequences, eg,first and second human Flt1 domain sequences; or first and second humanKDR domain sequences; or a Flt domain sequence and a KDR domainsequence. In an example, the trap comprises a human Flt1 Ig-like C2-type2 domain sequence and a human KDR Ig-like C2-type 3 domain sequence (eg,in this order in the N- to C-terminal direction).

In one embodiment, the ligand of the invention is a VEGF-A trapcomprising or of SEQ ID NO: 127, eg, comprising or consisting of a dimerof SEQ ID NO: 127.

In an example, the condition is an ophthalmic angiogenic disorder.

In an example, the condition is selected from the group consisting ofAMD, age-related macular degeneration; BRVO, branch retinal veinocclusion; CNV, choroidal neovascularization; mCNV, choroidalneovascularization caused by pathologic myopia; CRVO, central retinalvein occlusion; DME, diabetic macular edema; glaucoma; Macular edema;PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatousproliferation; retinal neovascularisation; ROP, retinopathy ofprematurity; and RVO, retinal vein occlusion.

In an example, the condition is exudative AMD.

In an example, the method is for the treatment of Adult-OnsetVitelliform Detachments Associated With Pattern Dystrophy.

In an example, the method is for the treatment of Pattern Dystrophy.

In an example, the method is for the treatment of Sub-Retinal Fibrosisin Neovascular AMD, eg, by administering Fovista® (an anti-PDGF BB) plusthe anti-VEGF ligand (eg, aflibercept, becavizumab or ranibizumab).

In an example, the human is aged ≧50 years, eg, the human is more than55, 60, 65 or 70 years of age.

In an example, the condition is an ocular condition and the anti-VEGF-Aligand is administered in combination with photodynamic therapy (PDT),eg, for treating or preventing PCV.

In an example, the condition is an ocular condition and the anti-VEGF-Aligand is administered in combination with laser treatment and/or IVTAtreatment, eg, for treating or preventing DME.

In an example, the condition is an ocular condition and the anti-VEGF-Aligand is administered in combination with laser photocoagulation and/orintravitreal injections of a glucocorticoid.

In an example, the condition is an ocular condition and the anti-VEGF-Aligand is administered in combination with PDT and triamcinoloneacetonide. Optionally, the ligand is administered intravitreally.

In an example the human is suffering from hypertension, hyperlipidemia,diabetes mellitus and/or arteriosclerotic vascular disorder. Optionally,the condition is RVO and further optionally the human is greater than 60years' of age.

In an example, when RVO is mentioned herein, the RVO is ischemic(non-perfusion) or non-ischemic (perfusion) RVO.

In an example, when ROP is mentioned herein, the type-1 ROP.

In an example, the condition is ROP and the anti-VEGF-A ligand isadministered in combination with laser treatment.

In an embodiment, the human has a Body Mass Index (BMI) ≧30 and/or thehuman is a cigarette smoker. These are risk factors, eg, wherein thedisease or condition is an ocular disease or condition.

Optionally there is less than 3 or 2 or 2 weeks between ocular diseaseor condition symptom onset and initiation of treatment with the ligandaccording to the invention.

In an example, the AMD is neovascular (nv) AMD, “wet” AMD or atrophic or“dry” AMD.

In an example, the ocular condition or disease (eg, AMD) is stage IVdisease according to the Age-Related Eye Disease Study classification(see, eg, Augood C A et al, “Prevalence of agerelated maculopathy inolder Europeans: the European Eye Study (EUREYE)”, Arch Ophthalmol.2006; 124:529e35) when the ligand is administered to the human.

In an embodiment, wherein the disease or condition is an ocular diseaseor condition (eg, AMD, PCV, DR or CNV), the ligand is administered tothe human by intraocular injection, eg, intravitreal injection.

Persistent angiogenesis may cause or exacerbate certain diseases such aspsoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabeticretinopathy and neovascular glaucoma. An inhibitor of VEGF activitywould be useful as a treatment for such diseases and other VEGF-inducedpathological angiogenesis and vascular permeability conditions, such astumor vascularization. Thus, in an embodiment the present invention inExample 5 provides a method (or ligand for use in the method) fortreating or reducing the risk of a condition associated withangiogenesis, wherein the condition is psoriasis, rheumatoid arthritis,hemangiomas, angiofibromas, diabetic retinopathy or neovascularglaucoma. In an embodiment the present invention in Example 5 provides amethod (or ligand for use in the method) for treating or reducing therisk of tumor vascularization.

By way of example, but not limitation, the method of the invention maybe useful in treating a clinical conditions that are characterized byvascular permeability, edema or inflammation such as brain edemaassociated with injury, stroke or tumor; edema associated withinflammatory disorders such as psoriasis or arthritis, includingrheumatoid arthritis; asthma; generalized edema associated with burns;ascites and pleural effusion associated with tumors, inflammation ortrauma; chronic airway inflammation; capillary leak syndrome; sepsis;kidney disease associated with increased leakage of protein; and eyedisorders such as age related macular degeneration and diabeticretinopathy. Thus, in an embodiment the present invention in Example 5provides a method (or ligand for use in the method) for treating orreducing the risk of any one of these conditions.

In an example, the method of the present invention comprisessequentially administering multiple doses of a VEGF antagonist to apatient. The methods of the present invention include the administrationof multiple doses of a VEGF antagonist to a patient at a frequency ofonce every 8 or more weeks.

Various administration routes are contemplated for use in the methods ofthe present invention, including, e.g., topical administration orintraocular administration (e.g., intravitreal administration). Theligand (or pharmaceutical formulation comprising the ligand) may beadministered to the patient by any known delivery system and/oradministration method. In certain embodiments, the ligand isadministered to the patient by ocular, intraocular, intravitreal orsubconjunctival injection. In other embodiments, the ligand can beadministered to the patient by topical administration, e.g., via eyedrops or other liquid, gel, ointment or fluid which contains the ligandand can be applied directly to the eye. Other possible routes ofadministration include, e.g., intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral.

The amount of anti-VEGF ligand administered to the patient in each doseis, in most cases, a therapeutically effective amount. As used herein,the phrase “therapeutically effective amount” means a dose of ligandthat results in a detectable improvement in one or more symptoms orindicia of an angiogenic eye disorder, or a dose of ligand thatinhibits, prevents, lessens, or delays the progression of an angiogeniceye disorder. In the case of an anti-VEGF antibody or a VEGFreceptor-based chimeric molecule (eg, VEGF trap, eg, aflibercept) suchas VEGFR1R2-FcΔC1(a), a therapeutically effective amount can be fromabout 0.05 mg to about 5 mg, e.g., about 0.05 mg, about 0.1 mg, about0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about0.9 mg, about 1.0 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg, about1.2 mg, about 1.25 mg, about 1.3 mg, about 1.35 mg, about 1.4 mg, about1.45 mg, about 1.5 mg, about 1.55 mg, about 1.6 mg, about 1.65 mg, about1.7 mg, about 1.75 mg, about 1.8 mg, about 1.85 mg, about 1.9 mg, about2.0 mg, about 2.05 mg, about 2.1 mg, about 2.15 mg, about 2.2 mg, about2.25 mg, about 2.3 mg, about 2.35 mg, about 2.4 mg, about 2.45 mg, about2.5 mg, about 2.55 mg, about 2.6 mg, about 2.65 mg, about 2.7 mg, about2.75 mg, about 2.8 mg, about 2.85 mg, about 2.9 mg, about 3.0 mg, about3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg of the antibody orreceptor-based chimeric molecule.

Dosing Regimens

Specific, non-limiting examples of dosing regimens within the scope ofthe present invention are as follows for an anti-TOI ligand (eg,anti-VEGF-A antibody or trap, eg, aflibercept):

-   -   a. ligand 2 mg (0.05 mL) administered by intravitreal injection        once every 4 weeks (monthly).    -   b. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05        mL) via intravitreal injection once every 8 weeks.    -   c. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05        mL) via intravitreal injection on a less frequent basis based on        visual and/or anatomical outcomes (as assessed by a physician or        other qualified medical professional).    -   d. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05        mL) via intravitreal injection administered pro re nata (PRN)        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   e. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 12 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection once every 8 weeks.    -   f. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 12 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection on a less frequent basis        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   g. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 12 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection administered pro re nata        (PRN) based on visual and/or anatomical outcomes (as assessed by        a physician or other qualified medical professional).    -   h. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 16 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection once every 8 weeks.    -   i. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 16 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection on a less frequent basis        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   j. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 16 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection administered pro re nata        (PRN) based on visual and/or anatomical outcomes (as assessed by        a physician or other qualified medical professional).    -   k. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 20 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection once every 8 weeks.    -   l. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 20 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection on a less frequent basis        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   m. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 20 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection administered pro re nata        (PRN) based on visual and/or anatomical outcomes (as assessed by        a physician or other qualified medical professional).    -   n. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 24 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection once every 8 weeks.    -   o. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 24 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection on a less frequent basis        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   p. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 24 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection administered pro re nata        (PRN) based on visual and/or anatomical outcomes (as assessed by        a physician or other qualified medical professional).    -   q. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 28 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection once every 8 weeks.    -   r. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 28 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection on a less frequent basis        based on visual and/or anatomical outcomes (as assessed by a        physician or other qualified medical professional).    -   s. ligand 2 mg (0.5 mL) administered by intravitreal injection        once every 4 weeks for the first 28 weeks, followed by 2 mg        (0.05 mL) via intravitreal injection administered pro re nata        (PRN) based on visual and/or anatomical outcomes (as assessed by        a physician or other qualified medical professional).    -   t. ligand 2 mg (0.05 mL) administered by intravitreal injection        as a single initial dose, followed by additional doses        administered pro re nata (PRN) based on visual and/or anatomical        outcomes (as assessed by a physician or other qualified medical        professional).

Variations on the above-described dosing regimens would be appreciatedby persons of ordinary skill in the art and are also within the scope ofthe present invention. For example, the amount of ligand and/or volumeof formulation administered to a patient may be varied based on patientcharacteristics, severity of disease, and other diagnostic assessmentsby a physician or other qualified medical professional.

When the method or ligand is for treating or reducing the risk of anocular condition or disease, the method in an example improves the BestCorrect Visual Acuity in the human.

Best Corrected Visual Acuity:

Visual function of the eye can be assessed using the ETDRS protocol (TheEarly Treatment Diabetic Retinopathy Study Group) at 4 meters. VisualAcuity examiners are typically certified to ensure consistentmeasurement of BCVA.

The method in an example method prevents vision loss of greater than orequal to 15 letters on the ETDRS chart, compared to baseline, at 52weeks. Additionally or alternatively the method provides one or more ofthe following: (a) change from baseline to Week 52 in letter score onthe ETDRS chart; (b) gain from baseline to Week 52 of 15 letters or moreon the ETDRS chart; (c) change from baseline to Week 52 in total NEIVFQ-25 score; and (d) change from baseline to Week 52 in CNV area (eg,where the condition is CNV).

In one embodiment, the ligand is an VEGF antagonist that comprises oneor more VEGF receptor-based chimeric molecule(s), (also referred toherein as a “VEGF-Trap” or “VEGFT”). An exemplary VEGF antagonist thatcan be used in the context of the present invention is a multimericVEGF-binding protein comprising two or more VEGF receptor-based chimericmolecules referred to herein as “VEGFR1R2-FcAC1(a)” or “aflibercept.”

Various ligand or antibody administration routes are contemplated foruse in the methods of the present invention, including, e.g., topicaladministration or intraocular administration (e.g., intravitrealadministration).

The methods of the invention optionally comprise sequentiallyadministering to a patient multiple doses of a ligand, eg, VEGFantagonist. As used herein, “sequentially administering” means that eachdose of ligand is administered to the patient at a different point intime, e.g., on different days separated by a predetermined interval(e.g., hours, days, weeks or months). The present invention includesmethods which comprise sequentially administering to the patient asingle initial dose of a ligand, followed by one or more secondary dosesof the ligand, followed by one or more tertiary doses of the ligand.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the ligand. Thus, the“initial dose” is the dose which is administered at the beginning of thetreatment regimen (also referred to as the “baseline dose”); the“secondary doses” are the doses which are administered after the initialdose; and the “tertiary doses” are the doses which are administeredafter the secondary doses. The initial, secondary, and tertiary dosesmay all contain the same amount of ligand, but will generally differfrom one another in terms of frequency of administration. In certainembodiments, however, the amount of ligand contained in the initial,secondary and/or tertiary doses will vary from one another (e.g.,adjusted up or down as appropriate) during the course of treatment.

Example 6 PD-L1 & PD-1

Application of PD-L1 Variation in the Present Invention

Human PD-L1 is also known as CD274, B7-H, B7H1, B7-H1, B7 homolog 1,MGC142294, MGC142296, PDCD1L1, PDCD1LG1, PDCD1 ligand 1, PDL1,Programmed cell death 1 ligand 1 and Programmed death ligand 1; and hasUniprot number Q9NZQ7 and NCBI gene ID number 29126. PD-L1 is a 290amino acid type I transmembrane protein encoded by the CD274 gene onhuman chromosome 9. PD-L1 expression is implicated in evasion of immuneresponses involved in chronic infection, e.g., by viruses (including,for example, HIV, HBV, HCV and HTLV, among others), by bacteria(including, for example, Helicobacter pylori, among others) and byparasites (including, for example, Schistosoma mansoni). CD274/PD-L1expression is also implicated in suppression of anti-tumour immuneactivity. Tumours express antigens that can be recognized by host Tcells, but immunologic clearance of tumours is rare. Part of thisfailure is due to immune suppression by the tumour microenvironment.PD-L1 expression on many tumours is a component of this suppressivemilieu and may act in concert with other immunosuppressive signals.PD-L1 expression has been shown in situ on a wide variety of solidtumours including breast, lung, colon, ovarian, melanoma, bladder,liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, andneck (Brown J A et al., 2003. J. Immunol. 170:1257-66; Dong H et al.2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad.Sci. USA 104:3360-65; Strome S E et al. 2003. Cancer Res. 63:6501-5;Inman B A et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin.Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol.Immunother. 56:1173-82; Nomi T et al. 2007. Clin. Cancer Res.13:2151-57; Thompson R H et al. 2004. Proc. Natl. Acad. Sci. USA101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang X G, Xu N. 2006. ActaHistochem. 108:19-24). In addition, PD-1 expression is upregulated ontumour infiltrating lymphocytes, and this may also contribute to tumourimmunosuppression (Blank C et al. 2003. J. Immunol. 171:4574-81). Inovarian cancer, PD-L1 expression is inversely correlated withintraepithelial, but not stromal, infiltrating CD8 T cells, suggestingthat PD-L1 inhibits the intratumor migration of CD8 T cells (Hamanishi Jet al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65). Translation ofPD-L1 mRNA is enhanced by loss of PTEN and the ensuing activation ofAkt, a common event in tumourigenesis (Parsa A T et al. 2007. Nat. Med.13:84-88). Most importantly, studies relating PD-L1 expression ontumours to disease outcome show that PD-L1 expression stronglycorrelates with unfavorable prognosis in kidney, ovarian, bladder,breast, gastric, and pancreatic cancer (Hamanishi J et al. 2007. Proc.Natl. Acad. Sci. USA 104:3360-65; Inman B A et al. 2007. Cancer109:1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100;Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82; Nomi Tet al. 2007. Clin. Cancer Res. 13:2151-57; Thompson R H et al. 2004.Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J,Zhang X G, Xu N. 2006. Acta Histochem. 108:19-24). In addition, thesestudies suggest that higher levels of PD-L1 expression on tumours mayfacilitate advancement of tumour stage and invasion into deeper tissuestructures.

The PD-1 pathway can also play a role in hematologic malignancies. PD-L1is expressed on multiple myeloma cells but not on normal plasma cells(Liu J et al. 2007.Blood 110:296-304). PD-L1 is expressed on someprimary T cell lymphomas, particularly anaplastic large cell T lymphomas(Brown J A et al., 2003. J. Immunol. 170:1257-66). PD-1 is highlyexpressed on the T cells of angioimmunoblastic lymphomas, and PD-L1 isexpressed on the associated follicular dendritic cell network (Dorfman DM et al. 2006. Am. J. Surg. Pathol. 30:802-10). In nodularlymphocyte-predominant Hodgkin lymphoma, the T cells associated withlymphocytic and/or histiocytic (L&H) cells express PD-1. Microarrayanalysis using a readout of genes induced by PD-1 ligation suggests thattumour-associated T cells are responding to PD-1 signals in situ inHodgkin lymphoma (Chemnitz J M et al. 2007. Blood 110:3226-33). PD-1 andPD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T cellleukemia and lymphoma (Shimauchi T et al. 2007. Int. J. Cancer 121:2585-90). These tumour cells are hyporesponsive to TCR signals.

Studies in animal models demonstrate that PD-L1 on tumours inhibits Tcell activation and lysis of tumour cells and in some cases leads toincreased tumour-specific T cell death (Dong H et al. 2002. Nat. Med.8:793-800; Hirano F et al. 2005. Cancer Res. 65:1089-96).Tumour-associated APCs can also utilize the PD-1:PD-L pathway to controlantitumor T cell responses. PD-L1 expression on a population oftumour-associated myeloid DCs is upregulated by tumour environmentalfactors (Curiel T J et al. 2003. Nat. Med. 9:562-67). Plasmacytoiddendritic cells (DCs) in the tumour-draining lymph node of B16 melanomaexpress IDO, which strongly activates the suppressive activity ofregulatory T cells. The suppressive activity of IDO-treated regulatory Tcells required cell contact with IDO-expressing DCs (Sharma M D et al.2007. J. Clin. Invest. 117:2570-82).

U.S. Pat. No. 8,507,663 discusses the specific inhibition of theexpression of the CD274/PD-L1 gene. Reference is also made toWO2011066389, WO2010036959, WO 2010077634, WO 03099196, WO 2013181634,WO2007005874, US20090317368 and U.S. Pat. No. 7,635,757.

The inventor carried out natural human variation in PD-L1 and identifiedthe following variations of interest for application to the presentinvention.

TABLE 21 Human PD-L1 Variation CHROMOSOMAL LOCATION AMINO AMINO SNP/(FORWARD NUCLEOTIDE ACID ACID CUMULATIVE VARIANT STRAND) CHANGE¹ CHANGE²POSITION³ FREQUENCY⁴ rs150439231 9: 5448539 rs138261640 9: 5448573rs142983488 9: 5448634 rs76741468 9: 5448641 rs822336 9: 5448690rs183400620 9: 5448763 rs187252832 9: 5448815 rs146143976 9: 5448859rs139023765 9: 5448896 rs73641615 9: 5448956 rs17718883 9: 5462876 P/R146 rs139709512 9: 5456126 A/P 5 rs140045210 9: 5462881 T/S 148rs141978642 9: 5466785 V/E 269 rs146495642 9: 5462929 G > A E/K 164rs370800260 9: 5457087 G > A V/I 21 rs143235887 9: 5457091 C > T T/M 22rs373692552 9: 5457117 G > C E/Q 31 rs140304675 9: 5457127 G > C S/T 34rs12551333 9: 5457171 G > C D/H 49 rs376993991 9: 5457282 C > T R/W 86rs367921713 9: 5462893 G > A E/K 152 rs41280721 9: 5462997 A > C R/S 186rs61752860 9: 5465514 A > T H/L 233 rs148141792 9: 5465595 G > A R/H 260rs369350813 9: 5467858 C > T T/M 290 rs10481593 9: 5458095 rs2282055 9:5455732 rs2297135 9: 5470108 rs2297136 9: 5467955 rs2297137 9: 5465732rs3780395 9: 5464552 rs7023227 9: 5456550 8923 A > C rs1411262 9:5459419 C > T 6777 C > G rs4143815 9: 5468257  395 G > C rs34028061 9:5454629-5454630 AT > — deletion 0.476 rs7041009 9: 5463243 G > A 0.459rs148170925 9: 5454642-5454643 TG > — deletion 0.600 rs1411262 9:5459419 C/T 0.395 rs10114060 9: 5461729 G/A 0.382 rs1536926 9: 5463988G/T 0.360 rs7042084 9: 5458035 G/T 0.352 ^(1,2,3,4)According to Ensembl,dbSNP; with reference to transcript ENST00000381577 (the sequence ofthis transcript being incorporated herein in its entirety).

The table is to read as follows: For example, the following forrs12551333 means Asp49His, ie, a histidine at position 49 encoded by SNPrs12551333

References

-   1. Asia Pac J Clin Oncol. 2014 June; 10(2):e1-6. doi:    10.1111/ajco.12037. Epub 2012 Nov. 21;    “Association between single nucleotide polymorphism of PD-L1 gene    and non-small cell lung cancer susceptibility in a Chinese    population”; Chen Y et al.    The authors published the following:—    Aim:    To evaluate the correlation between a polymorphism of PD-L1 gene and    the susceptibility of non-small cell lung cancer (NSCLC) in a    Chinese population.    Methods: A total of 293 Chinese patients with NSCLC and 293 age and    sex matched controls of the same ethnic origin were enrolled in this    study. A/C polymorphism at position 8923 in intron 4 of PD-L1 gene    was typed using the polymerase chain reaction-restriction fragment    length polymorphism method (PCR-RFLP). The interactions between A/C    genotype, allele frequency and NSCLC susceptibility were analyzed.    Results:    The A/C genotype frequencies were significantly different between    NSCLC patients and controls. The AC and CC frequencies were higher    in NSCLC patients than in controls (16.4 vs 8.9%, 1.0 vs 0.3%,    respectively). The C-allele frequency was higher in NSCLC patients    than in controls (9.2 vs 4.8%). Significant differences in the A and    C allele frequencies were noted between the two groups (χ(2)=8.864,    P=0.003). More risk of NSCLC was found in individuals carrying the C    allele than in those carrying the A allele (OR=2.203; 95% CI    1.262-3.242). In both light smokers (<20 pack-years) and heavy    smokers (>20 pack-years), individuals carrying the C-allele had more    risk of NSCLC than those carrying the A-allele (light smokers    OR=1.847, 95% CI 1.001-3.409; heavy smokers OR=3.252, 95% CI    1.196-8.845, respectively).    Conclusion:    An A/C polymorphism at position 8923 in the PD-L1 gene is associated    with NSCLC susceptibility. The PD-L1 polymorphism plays a role in    NSCLC, especially in patients with the C-allele.-   2. Eur J Endocrinol. 2008 June; 158(6):817-22. doi:    10.1530/EJE-07-0649. Epub 2008 Mar. 5.    “Association of an A/C single nucleotide polymorphism in programmed    cell death-ligand 1 gene with Graves' disease in Japanese patients”;    Hayashi M et al.    The authors published the following:—    Objective:    Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2)    inhibit T-cell proliferation and activation. This inhibition    down-regulates the immune responses. The association of a PD-L1    polymorphism with Graves' disease (GD) was studied.    Design:    The association of an A/C polymorphism at position 8923 in PD-L1    intron 4 with GD was studied.    Patients:    The study included 327 GD patients and 192 controls, of which 252 GD    patients were followed over 5-10 years.    Measurements:    PD-L1 intron 4 position 8923 A/C polymorphism was typed using the    PCR-restriction fragment length polymorphism method.    Results:    The A/C genotype frequencies were significantly different between GD    patients and controls. The A/C and C/C frequencies were higher in GD    patients than in controls. The A/A frequencies were lower in GD    patients than in controls. C-allele frequency was higher in GD    patients than in controls. A total of 252 GD patients were followed    over 5-10 years; 200 had discontinued antithyroid drugs (ATD) while    52 continued to take ATD. Of these 200, 176 continued to be in    remission and 24 had relapsed into hyperthyroidism. Significant    differences in the duration of positive TBII, positive    thyroid-stimulating antibodies, and ATD treatment were noted between    the patients in remission and those that had relapsed. Significant    differences in the A- and C-allele frequencies were noted between    the two. The C-allele frequency was higher in GD patients who did    not achieve remission than in those who achieved remission.    Conclusion:    An A/C polymorphism at position 8923 in PD-L1 is associated with GD.    The PD-L1 polymorphism plays a role in GD development. GD patients    with the C allele at position 8923 in PD-L1 gene had difficulty in    achieving remission.-   3. Rheumatology (Oxford). 2011 October; 50(10):1809-13. doi:    10.1093/rheumatology/ker211. Epub 2011 Jul. 26; “Effects of genetic    polymorphisms of programmed cell death 1 and its ligands on the    development of ankylosing spondylitis”; Huang C et al.    Human subjects with the PD-1 GG genotype had significantly greater    risk for ankylosing spondylitis (AS) than those with the AA    genotype. Subjects with the PD-L2 CT genotype had lower risk for AS    than those with the CC genotype. The combined genotypes of PD-1    G-536A, PD-L1 A8923C and PD-L2 C47103T also appear to be associated    with AS development.    Conclusions.    The results suggest that PD-1 G-536A, PD-L1 A8923C and PD-L2 C47103T    polymorphisms are associated with the presence of AS.-   4. J Clin Endocrinol Metab. 2009 December; 94(12):5139-45. doi:    10.1210/jc.2009-1404. Epub 2009 Oct. 22; “Programmed death ligand 1    (PD-L1) gene variants contribute to autoimmune Addison's disease and    Graves' disease susceptibility”; Mitchell A L et al.    The authors published the following:—    Context:    Despite much investigation, a substantial amount of the genetic    susceptibility to autoimmune diseases remains unaccounted for.    Recently, a single-nucleotide polymorphism (SNP) in the programmed    death ligand 1 (PD-L1) gene has been associated with Graves' disease    (GD) in a Japanese patient cohort. Our aim was to determine whether    variants in PD-L1 are also associated with autoimmune Addison's    disease (AAD) and to replicate the previous association in patients    with GD from the United Kingdom.    Design and Patients:    We analyzed eight SNPs within PD-L1 in a United Kingdom cohort of    315 AAD subjects and 316 healthy controls. We then replicated our    experiment in a cohort of 342 Norwegian AAD cases and 379 controls    and in 496 United Kingdom GD subjects.    Results:    Three of the eight SNPs studied, part of a haplotype block in the    PD-L1 gene, showed modest association with both AAD and GD in the    United Kingdom cohort, with maximum evidence at the marker RS1411262    [United Kingdom AAD odds ratio 1.33 (5-95% confidence interval    1.02-1.73), P(genotype)=0.028; GD odds ratio 1.36 (5-95% confidence    interval 1.07-1.72), P(genotype)=0.033]. Association with genotypes    at the same three markers was confirmed in the Norwegian AAD cohort    [P(genotype)=0.011-0.020]. A recessive effect at the most associated    alleles was observed in both the AAD and GD cohorts.    Conclusions:    We confirm the role of PD-L1 variants in GD susceptibility and    extend these findings to demonstrate association in two Northern    European patient cohorts with AAD. PD-L1 joins the growing number of    known susceptibility loci exerting modest effects in these    autoimmune disorders.    5. J Clin Immunol. 2007 November; 27(6):563-7. Epub 2007 Jun. 28;    “Polymorphisms of genes for programmed cell death 1 ligands in    patients with rheumatoid arthritis”; Wang S C et al.    The authors published the following:—    To investigate the role of ligands for programmed cell death 1    (PD-L) in the pathogenesis of rheumatoid arthritis (RA), 129    patients with RA and 125 unrelated healthy controls were enrolled in    this study. The PD-L1 and PD-L2 polymorphisms were determined by the    method of polymerase chain reaction (PCR)/direct sequencing or    PCR/reaction fragment length polymorphisms. The genotype    distributions of PD-L1 6777 C/G were not significantly different    between the patients with RA and healthy controls. There was also no    significant difference in the allele frequencies of PD-L1 6777 C/G    polymorphisms between the patients with RA and controls. Similar    findings could also be found in the phenotypes and alleles    frequencies of PD-L2 47103 C/T and 47139 T/C polymorphisms between    the patients with RA and controls. The patients with PD-L1 6777 G    had higher prevalence of rheumatoid nodule in comparison with those    without PD-L1 6777 G (p=0.005, OR=4.0, 95% CI=1.5-10.9). In    contrast, the PD-L2 47103 C/T and 47139 T/C polymorphisms were not    related to the occurrence of rheumatoid nodule. This study    demonstrated that the PD-L1 and PD-L2 polymorphisms were not    associated with susceptibility to RA in Taiwan. PD-L1 6777 G was    associated with the prevalence of rheumatoid nodule.-   6. Hum Genet. 2013 June; 132(6):641-8. doi:    10.1007/s00439-013-1275-6. Epub 2013 Feb. 21; “A miR-570 binding    site polymorphism in the B7-H1 gene is associated with the risk of    gastric adenocarcinoma”; Wang W et al.    Since the over-expression of B7-H1 protein has been reported to be    closely related to disease progression of gastric cancer, the    authors investigated the possible role of miRSNPs at the    3′-untranslated region (3′-UTR) of B7-H1 in the risk of developing    gastric cancer. In this association study on 205 gastric    adenocarcinoma patients and 393 non-cancer controls, they found that    the genotype distribution of a common C>G polymorphism (rs4143815)    was significantly different between the cases and controls    (P=1.32×10(−8)). Compared with CC homozygotes, GG homozygotes and G    allele carriers showed 3.73-fold (P=2.98×10(−8)) and 1.85-fold    (P=0.002) increased risk of gastric adenocarcinoma, respectively.    Stratified analyses indicated that variant genotypes had a strong    association with the clinic-pathological features of gastric cancer    including differentiation grade, depth of tumor infiltration, and    tumor node metastasis (TNM) stage (P<0.001). Luciferase reporter    assay indicated that this SNP might be responsible for aberrant    B7-H1 protein expression in gastric cancer by disrupting the    interaction between miR-570 and B7-H1 mRNA. These results are    consistent with our hypothesis and indicate that genetic    polymorphisms influencing B7-H1 expression modify cancer    susceptibility.-   7. Clinical cancer research: an official journal of the American    Association for Cancer Research/15(3):971-9; Pub Date: Feb. 1, 2009;    “Overexpression of PD-L1 significantly associates with tumor    aggressiveness and postoperative recurrence in human hepatocellular    carcinoma”; Qiang G et al.    The authors published the following:—    PURPOSE The aberrant expression of programmed cell death 1 ligands 1    and 2 (PD-Ls) on tumor cells dampens antitumor immunity, resulting    in tumor immune evasion. In this study, we investigated the    expression of PD-Ls in human hepatocellular carcinoma (HCC) to    define their prognostic significance after curative surgery.    EXPERIMENTAL DESIGN Immunohistochemistry was used to investigate    PD-Ls expression as well as granzyme B+ cytotoxic and FoxP3+    regulatory T cell infiltration on tissue microarrays containing 240    randomly selected HCC patients who underwent surgery. The results    were further verified in an independent cohort of 125 HCC patients.    PD-Ls expression on HCC cell lines was detected by Western blot    assay.    RESULTS Patients with higher expression of PD-L1 had a significantly    poorer prognosis than patients with lower expression. Although    patients with higher expression of PD-L2 also had a poorer survival,    the difference in recurrence was not statistically significant.    Multivariate analysis identified tumor expression of PD-L1 as an    independent predictor for postoperative recurrence. No correlation    was found between PD-Ls expression and granzyme B+ lymphocyte    infiltration, whereas a significant positive correlation was    detected between PD-Ls expression and FoxP3+ lymphocyte    infiltration. In addition, tumor-infiltrating cytotoxic and    regulatory T cells were also independent prognosticators for both    survival and recurrence. The prognostic value of PD-L1 expression    was validated in the independent data set.    CONCLUSION Our data suggest for the first time that PD-L1 status may    be a new predictor of recurrence for HCC patients and provide the    rationale for developing a novel therapy of targeting the PD-L1/PD-1    pathway against this fatal malignancy.

Using this analysis, the inventor has devised the following aspects ofthe invention that are useful for addressing PD-L1-mediated diseases andconditions, such as cancer, autoimmune or inflammatory diseases andconditions as more fully described below. Thus, the invention providesthe following aspects.

In an example, the invention provides a method of treating or reducingthe risk of a PD-L1-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human PD-L1protein. The invention also provides a corresponding ligand. In anembodiment, the PD-L1 is encoded by a PD-L1 nucleotide sequencecomprising a variation set out in Table 21, eg, comprising one or moreof said SNPs (eg, RS1411262 and/or rs4143815; or 8923C and/or 395C). Ina preferred embodiment, the variation is 8923C.

The present invention provides anti-PD-L1 ligands; and PD-L1-binding ortargeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of PD-L1, in particular human PD-L1 or its ligands and inscreening assays to identify other antagonists of PD-L1 activity. Someof the ligands of the invention are useful for inhibiting PD-L1-mediatedactivities.

Anti-PD-L1 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human PD-L1, which is a commonly-occurring form. While such therapiesare useful for human patients harbouring this form of human PD-L1, theinventor considered it useful to investigate the possibility oftargeting rarer—but still naturally-occurring—forms of PD-L1 amongsthuman populations. In this way, the inventor arrived at insight into thenatural occurrences and distributions of rarer human PD-L1 forms thatcan serve as useful targets (at the protein or nucleic acid level) forhuman treatment, prophylaxis and diagnosis pertinent to diseases andconditions mediated or associated with PD-L1 activity. This particularlyprovides for tailored therapies, prophylaxis and diagnosis in humansthat are devoid of the common PD-L1 gene or protein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer PD-L1 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous (aka“missense”) changes at the nucleotide level that translate into one ormore corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that the rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting such rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention. In particular, humanPD-L1 variations are correlated with increased incidence or risk ofPD-L1 mediated diseases or conditions, such as cancer. The inventoranalysed such variations and devised the collections of variants inTable 21. In this respect, therefore, the invention provides variousaspects and aspects as set out below in this example.

The inventor saw that there is significant industrial and medicalapplication for the invention in terms of guiding the choice of anti-TOI(PD-L1) ligand for administration to human patients for therapy and/orprophylaxis of PD-L1-mediated or associated diseases or conditions. Inthis way, the patient receives drugs and ligands that are tailored totheir needs—as determined by the patient's genetic or phenotypic makeup.Hand-in-hand with this, the invention provides for the genotyping and/orphenotyping of patients in connection with such treatment, therebyallowing a proper match of drug to patient. This increases the chancesof medical efficacy, reduces the likelihood of inferior treatment usingdrugs or ligands that are not matched to the patient (eg, poor efficacyand/or side-effects) and avoids pharmaceutical mis-prescription andwaste.

In developing this thinking, in a non-limiting embodiment the presentinventor decided to determine a set of human PD-L1 variants on the basisof the following criteria, these being criteria that the inventorrealised would provide for useful medical drugs and diagnostics totailored need in the human population. In an embodiment the inventorselected variants having at least 3 of the 4 following criteria:—

-   -   Naturally-occurring human TOI variation having a cumulative        human allele frequency of 49 or 35% or less;    -   Naturally-occurring human TOI variation having a total human        genotype frequency of about 50% or less;    -   Naturally-occurring human TOI variation found in many different        human ethnic populations (using the standard categorisation of        the 1000 Genomes Project; see Table 2 below); and    -   Naturally-occurring human TOI variation found in many        individuals distributed across such many different ethnic        populations.

In an example, the ligand of the invention comprises an anti-human PD-L1binding site, wherein the binding site is a human or humanized bindingsite, eg, the binding site comprises or consists of a human or humanizedantibody variable domain or plurality of variable domains (eg, humanVH/VL binding site(s)). Additionally or alternatively, the ligandcomprises one or more human antibody constant regions (eg, a humanantibody CH1, CH2, CH3 (or all of these) or Fc). In an example, theligand is an antibody that comprises human or humanized variable regionsand human constant regions (eg, bearing one or more mutations to enhanceor dampen Fc function in a human patient).

In an example, the invention provides a method of targeting PD-L1 in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human PD-L1protein that is encoded by a human PD-L1 nucleotide comprising a PD-L1variation as described herein. In an example, the human is sufferingfrom or at risk of a PD-L1-mediated disease or condition. In an example,the method treats or reduces the risk of a PD-L1-mediated disease orcondition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.

Preferably, additionally or alternatively in an embodiment theanti-PDL-1 ligand (eg, antibody or fragment) comprises a gamma-1constant region (eg, an IGHG1*01 constant region) as described herein.

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73.

In a specific embodiment, the anti-PD-L1 ligand, antibody or fragment ofpresent invention comprises an Fc region, wherein the Fc regioncomprises at least one non-native amino acid residue selected from thegroup consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341,234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H,235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H,239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R. 243W, 243L243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L,256E, 256M, 262I, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 264I,264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F,265V, 2651, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E,269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D,296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 298H, 2981,298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F,316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W,327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T,328H, 328A, 329F, 329H, 329Q, 330K, 330G, 3301, 330C, 330L, 330Y, 330V,3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q,331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D,332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E,370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W as numbered bythe EU index as set forth in Kabat. Optionally, the Fc region maycomprise additional and/or alternative non-native amino acid residuesknown to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919;WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

The ligand, antibody or fragment according to the invention (eg, ananti-PD-L1 antibody or fragment) is for treating or preventing orreducing the risk of (or treats or prevents or reduces the risk of), forexample, any disease or condition disclosed in U.S. Pat. No. 8,507,663,WO2011066389, WO2010036959, WO 2010077634, WO 03099196, WO 2013181634,WO2007005874, US20090317368 or U.S. Pat. No. 7,635,757, the disclosureof which diseases and conditions are incorporated herein by referencefor potential inclusion in one or more aspects herein. Guidance onobtaining and testing antibodies can also be found in thesepublications. Suitable anti-PD-L1 ligands, antibodies or fragments foruse in the invention are disclosed in these publications, the disclosureof which (including sequences thereof) are incorporated herein byreference for potential inclusion in one or more aspects herein.

Further encompassed by the invention is the use of the anti-PD-L1ligand, antibody or fragment in the manufacture of a medicament for useto attenuate or inhibit a PD-L1-mediated disease or disorder in a human.PD-L1-mediated or related disorders which are treated by the ligand,antibody or fragment of the invention include, for example, describedbelow.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving PD-L1expression and/or activity. One embodiment, among others, is a method oftreatment comprising administering an effective amount of a ligand,antibody or fragment of the invention to a patient in need thereof,wherein functional consequences of PD-L1 activation are decreased.Another embodiment, among others, is a method of treatment comprising(i) identifying a patient demonstrating PD-L1 expression or activity,and (ii) administering an effective amount of a ligand, antibody orfragment of the invention to the patient, wherein a functionalconsequence of PD-L1 activation are attenuated. An effective amountaccording to the invention is an amount that modulates (e.g. decreases)the functional consequences of PD-L1 activation so as to modulate (e.g.decrease or lessen) the severity of at least one symptom of theparticular disease or disorder being treated, but not necessarily curethe disease or disorder. Accordingly, one embodiment of the invention isa method of treating or reducing the severity of at least one symptom ofany of the disorders referred to herein, comprising administering to apatient in need thereof an effective amount of one or more ligands,antibodies or fragments of the present invention alone or in a combinedtherapeutic regimen with another appropriate medicament known in the artor described herein such that the severity of at least one symptom ofany of the disorders is reduced. Another embodiment of the invention,among others, is a method of antagonizing at least one effect of PD-L1comprising contacting with or administering an effective amount of oneor more ligands, antibodies or fragments of the present invention suchthat said at least one effect of PD-L1 is antagonized, e.g. the abilityto promote or maintain cancer, an autoimmune condition or aninflammatory condition.

Tailoring Anti-PD-L1 Ligands

As outlined herein (for example, in the context of PCSK9 in Example 1),the invention includes the possibility to tailor treatment of humansfurther by selecting antibody-based ligands with variable domains and/orconstant domains based on gene segments found in many humans of theethnic populations where the variant TOI forms are found to meet theselection criteria of the invention. This also applies mutatis mutandiswhere the TOI is human PD-L1 as in the present example. Thus, alldisclosure herein relating to tailoring variable and/or constant domainsapply to the present example, relating to PD-L1 and is combinable foruse in one or more aspects herein.

As described in Example 1, an example is provided for ligands comprisingantibody VH domains derived from recombination of human IGHV genesegments comprising selected nucleotides at positions in the HCDR1 orFW3 where there is variability in humans (ie, where SNPs occur inhumans).

Further information is provided in Table 4, which shows variation atthese positions, as well as the variant distributions across the 1000Genomes Project database relating to many human populations.

In other embodiments, as explained more fully above, the inventionprovides for ligands which are tailored to the human recipient'sgenotype and/or phenotype based on alternative human VH gene segments,or on Vκ, Vλ, or constant region gene segments (see further Table 9 forrepresentative variants).

Further examples, therefore are:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment, and wherein said human comprises a IGHV3-7*01 VHgene segment or the human expresses VH domains derived from therecombination of human VH segment IGHV3-7*01, a human D gene segment anda human JH segment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment IGKV1-12*01 and a human Jκ segment,and wherein said human comprises a IGKV1-12*01 Vκ gene segment or thehuman expresses Vκ domains derived from the recombination of human Vκsegment IGKV1-12*01 and a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4.(vi) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Valat position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shownin SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asnat position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ IDNO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257shown in SEQ ID NO: 6. In an example, the constant region is Fcenhanced, eg, ADCC or CDC enhanced. The skilled person will knowtechniques (eg, known C region mutations) to enhance ADCC or CDC.(vii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(viii) wherein the ligand comprises a human IGLC1*01 lambda chainconstant region and wherein said human comprises (i) a human IGLC1*01lambda chain constant region gene segment, or the human expressesantibodies comprising human IGLC1*01 lambda chain constant regions.(ix) wherein the ligand comprises a human gamma-4 heavy chain constantregion that comprises a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73 and wherein said humancomprises (i) an IGHG4*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-4heavy chain constant regions comprising a Leu at position 189 shown inSEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73.(x) wherein the ligand comprises a human gamma-3 heavy chain constantregion encoded by a first human IGHG3 (eg, IGHG3*01) constant regiongene segment and wherein said human comprises (i) said first constantregion gene segment (eg, an IGHG3*01), or the human expresses antibodiescomprising human gamma-3 heavy chain constant regions encoded by saidfirst human IGHG3 (eg, IGHG3*01) constant region gene segment.(xi) wherein the ligand comprises a human epsilon heavy chain constantregion encoded by a first human epsilon heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human epsilonheavy chain constant regions encoded by said first constant region genesegment.(xii) wherein the ligand comprises a human mu heavy chain constantregion encoded by a first human mu heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human muheavy chain constant regions encoded by said first constant region genesegment.(xiii) wherein the ligand comprises a human alpha heavy chain constantregion encoded by a first human alpha heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human alphaheavy chain constant regions encoded by said first constant region genesegment.(xiv) wherein the ligand comprises a human delta heavy chain constantregion encoded by a first human delta heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human deltaheavy chain constant regions encoded by said first constant region genesegment.(xv) wherein the ligand comprises a human kappa light chain constantregion encoded by a first human kappa light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human kappalight chain constant regions encoded by said first constant region genesegment.(xvi) wherein the ligand comprises a human lambda light chain constantregion encoded by a first human lambda light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human lambdalight chain constant regions encoded by said first constant region genesegment.

Generally, in an example the ligand comprises a heavy chain constantregion that is Fc enhanced, eg, ADCC or CDC enhanced. The skilled personwill know techniques (eg, known C region mutations; defucosylation toenhance ADCC) to enhance ADCC or CDC. This applies to any of Examples 1onwards herein.

Thus, it may be advantageous for PD-L I ligands, for the ligand tocomprise a gamma-1 or gamma-2 constant region (eg, as per theembodiments (i) to (xvi) above). For example, the ligand comprises saidgamma-1 constant region, eg, an ADCC or CDC enhanced constant regionmeeting embodiment (v).

In an example, the ligand (eg, an antibody) is conjugated to a cytokineselected from the group consisting of IL-2, IL-12, IL-15 or IL-21. In anexample, the cytokine is IL-2.

In an example, eg, for where the condition is a viral infection, theligand (eg, antibody) comprises a gamma-4 constant region as hereindefined and optionally wherein an Fc inactivated IgG4, such aIgG4(S228P).

Determination of Specific Binding of Ligands of the Invention to PD-L1Variants

The specific binding of ligands of the invention to PD-L I variants canbe performed using the SPR method described in Example 1.

By way of example, the invention thus provides the following aspects:—

1. A method of treating or reducing the risk of a disease or conditionmediated by PD-L I in a human, the method comprising administering tosaid human an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody orantibody fragment) that specifically binds to a human PD-L1 that isexpressed by a PD-L1 nucleotide sequence comprising a variation selectedfrom the variations listed in Table 21.For example, there is provided a method of treating or reducing the riskof a disease or condition mediated by PD-L1 in a human, the methodcomprising administering to said human an anti-PD-L1 ligand (eg, ananti-PD-L1 trap, antibody or antibody fragment) that specifically bindsto a human PD-L1 that is expressed by a PD-L1 nucleotide sequencecomprising a variation selected from the variations listed in Table 21,wherein said human comprises a PD-L1 nucleotide sequence comprising saidselected variation.For example, there is provided a method of treating or reducing the riskof a cancer in a human, the method comprising administering to saidhuman an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibodyfragment) that specifically binds to a human PD-L1 that is expressed bya PD-L1 nucleotide sequence comprising a variation selected from thevariations listed in Table 21, wherein said human comprises a PD-L1nucleotide sequence comprising said selected variation.For example, there is provided a method of treating or reducing the riskof an autoimmune disease or condition in a human, the method comprisingadministering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1trap, antibody or antibody fragment) that specifically binds to a humanPD-L1 that is expressed by a PD-L1 nucleotide sequence comprising avariation selected from the variations listed in Table 21, wherein saidhuman comprises a PD-L1 nucleotide sequence comprising said selectedvariation.For example, there is provided a method of treating or reducing the riskinflamatory disease or condition in a human, the method comprisingadministering to said human an anti-PD-L1 ligand (eg, an anti-PD-L1trap, antibody or antibody fragment) that specifically binds to a humanPD-L1 that is expressed by a PD-L1 nucleotide sequence comprising avariation selected from the variations listed in Table 21, wherein saidhuman comprises a PD-L1 nucleotide sequence comprising said selectedvariation.Herein, specific binding can be tested using a sample PD-L1 using aroutine method known the skilled person, eg, ELISA or SPR (eg, using amethod as described herein). For example, the PD-L1 is provided by asample from said human or another human, eg, a serum or blood sample.In an example, the ligand comprises an anti-PD-L1 binding site thatspecifically binds human PD-L1. Optionally, the binding site comprisesone or more antibody variable domains or one or more human PD-L1receptor domains. In an example, specific binding with a Kd of 1 mm orless (ie, 1 mM or stronger binding), eg, 1 nM or less; or 100 pM orless; or 10 pM or less. Specific binding can be determined, for example,by assessing binding of the ligand to PD-L1 in a sample from said humanor another human comprising said PD-L1. In an example, the samplecomprises serum, blood, faeces, tissue, a cell, urine and/or saliva ofsaid human.In an example, the ligand is selected from the group consisting of wherMEDI4736 (Medimmune), durvalumab, RG-7446 (Genentech, Roche, Chugai,MPDL3280A), MPDL3280A (Genentech), STI-A1014 (Sorrento TherapeuticsInc), MSB-0010718C (Merck Serono); MDX1105 (Bristol-Myers Squibb) andBMS-936559 (Bristol-Myers Squibb). Optionally the ligand is administeredin combination with an anti-CTLA-4 ligand (eg, antibody or fragment),eg, Tremelimumab. For example, MEDI4736 or durvalumab is administered incombination with an anti-CTLA-4 ligand (eg, antibody or fragment), eg,Tremelimumab.In an example, the ligand is an anti-PD-L1 antibody or antibodyfragment, eg, durvalumab.In an example, the ligand is an NCE (so-called New Chemical Entity inthe art).In an example, the human has been diagnosed with said disease orcondition before said administration of the ligand.For example, the human is partially or completely resistant to treatmentwith an anti-PD-L1 ligand, eg, an anti-PD-L1 NCE.In an example, the ligand is administered by intravenous or subcutaneousadministration and/or is comprised in an injectable preparation.2. The method of aspect 1, wherein the nucleotide sequence comprises anucleotide corresponding to SNP rS 1411262, SNP rs4143815, 8923C or395C.3. The method of aspect 1 or 2, wherein said human comprises a PD-L1nucleotide sequence comprising said selected variation.Optionally, the human is homozygous for said variation. Alternatively,the human is heterozygous for said variation.In an example, the ligand comprises an antibody constant region (eg, anantibody Fc region).4. The method of any one of aspects 1 to 3, wherein the ligand comprisesa human gamma-1 heavy chain constant region that comprises an amino acidselected from the group consisting of an Asp corresponding to position204 of SEQ ID NO: 42 and a Leu corresponding to position 206 of SEQ IDNO: 42 and optionally wherein said human comprises an IGHG1*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-1 heavy chain constant regionscomprising said selected amino acid.For example, the ligand comprises a human gamma-1 heavy chain constantregion that comprises an amino acid selected from the group consistingof an Asp corresponding to position 204 of SEQ ID NO: 42 and a Leucorresponding to position 206 of SEQ ID NO: 42 and wherein said humancomprises an IGHG1*01 human heavy chain constant region gene segment, orthe human expresses antibodies comprising human gamma-1 heavy chainconstant regions comprising said selected amino acid. In an embodiment,this is combined with any of the examples set out under aspect 1 above.Optionally, this constant region of the ligand is comprised by an Fcregion, eg, an enhanced Fc region.Optionally, the ligand comprises an IGHG1*01 human heavy chain constantregion.5. The method of any one of aspects 1 to 4, wherein the disease orcondition is cancer (eg, a solid tumour), an autoimmune disease orcondition; or an inflammatory disease or condition. In an example, thecancer is selected from the group consisting of breast cancer, lungcancer (eg, non-small cell lung cancer), colon cancer, ovarian cancer,melanoma (eg, stage IV melanoma), bladder cancer, liver cancer (eg,hepatocellular carcinoma), kidney cancer, salivary gland cancer, stomachcancer, gastric cancer, glioma, pancreas cancer, thyroid cancer, thymicepithelial cancer, head cancer and/or neck cancer, multiple myeloma, Tcell lymphoma, Hodgkin lymphoma, renal cell carcinoma, cervical cancer,colorectal, a haematological neoplasm, metastatic colorectal cancer,uterine or cervix cancer, a leukaemia (eg, acute myelogenousleukaemia),diffuse large B-cell lymphoma, follicle centre lymphoma,prostate cancer and Merkel cell carcinoma.Alternatively, the disease or condition is a viral infection, eg, HIV,hepatitis C or Ebola infection. Alternatively, the disease or conditionis melodysplastic syndrome.In an example, the condition is a cancer, eg, metastatic cancer.6. The method of any one of aspects 1 to 5, comprising, before saidadministering, selecting a human comprising said PD-L1 nucleotidesequence, wherein the human is the human of aspect 1.7. The method of any one of aspects 1 to 6, wherein the human has beendetermined to comprise said PD-L1 nucleotide sequence.8. The method of any one of aspects 1 to 6, comprising the step ofdetermining that the human comprises said PD-L1 nucleotide sequence,optionally, wherein the determining step is performed beforeadministration of the antibody to the human.9. The method of aspect 8, wherein the step of determining comprisesassaying a biological sample from the human for a PD-L1 nucleotidesequence comprising said selected variation.10. The method of aspect 9, wherein the assaying comprises contactingthe biological sample with

-   -   a. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence comprising said selected variation or that specifically        hybridizes to an antisense of said sequence, wherein said        nucleic acid hybridizes to said selected variation or hybridizes        to an antisense sequence thereby forming a complex when at least        one nucleotide sequence comprising said selected variation is        present; and/or    -   b. at least one oligonucleotide probe comprising a sequence of        at least 10 contiguous nucleotides of a nucleotide sequence        comprising said selected variation or comprising an antisense        sequence of said contiguous nucleotides, wherein said sequence        of contiguous nucleotides comprises said selected variation        thereby forming a complex when the nucleotide sequence        comprising said selected variation is present; and detecting the        presence or absence of the complex, wherein detecting the        presence of the complex determines that the human comprises a        PD-L1 nucleotide sequence comprising said selected variation.        11. The method of aspect 10, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.        12. The method of any one of aspects 1 to 11, wherein said human        is or has been further determined to be substantially resistant        a PD-L1 or PD-1 treatment.        13. The method of any one of aspects 1 to 11, wherein said human        is receiving or has received a PD-L1 or PD-1 treatment or has        reduced responsiveness to a PD-L1 or PD-1 treatment.        14. The method of aspect 9, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.        15. The method of any one of aspects 1 to 14, wherein said human        has been diagnosed with a cancer, autoimmune disease or        condition; or an inflammatory disease or condition.        16. The method of any one of aspects 1 to 15, wherein said        ligand is administered by intravenous or subcutaneous injection        and/or is comprised in an injectable preparation.        17. The method of any one of aspects 1 to 16, wherein the ligand        is human.        18. An anti-human PD-L1 ligand (eg, an antibody, antibody        fragment or human PD-L1 trap) for use in a method of any one of        aspects 1 to 17.        19. The ligand of aspect 18, wherein the ligand is a human        anti-human PD-L1 antibody (eg, durvalumab), antibody fragment or        human PD-L1 trap.        20. The ligand of aspect 18 or 19, wherein said ligand is for        intravenous or subcutaneous administration and/or is comprised        in an injectable preparation.

For rs148170925, the 1000 Genomes Phase 1 database indicates acumulative frequency across all human populations of 60% for thedeletion (ie, presence on the SNP) and 40% for absence of the SNP (ie,presence of TG at chromosomal location 9:5454642-5454643). The followinghuman populations have above average frequency for absence of the SNP:AMR (45%), EUR (54%), ASW (44%), CLM (42%), MXL (44%), PUR (49%), CEU(60%), FIN (57%), GBR (56%), IBS (46%) and TSI (46%); in an embodimentof the invention, the human is of any one of these ancestries. Thefollowing human populations have above average frequency for presence ofthe SNP: AFR (67%), ASN (76%), LWK (77%), YRI (65%), CHB (73%), CHS(79%), JPT (75%); in an embodiment of the invention, the human is of anyone of these ancestries.

Example 7 TOIs For Cancer, Autoimmune & Inflammatory ApplicationsExample 7A Tumour Targeting & Immuno-Oncology Applications

The tumour microenvironment is an important aspect of cancer biologythat contributes to tumour initiation, tumour progression and responsesto therapy. Cells and molecules of the immune system are a fundamentalcomponent of the tumour microenvironment. Importantly, therapeuticstrategies can harness the immune system to specifically target tumourcells and this is particularly appealing owing to the possibility ofinducing tumour-specific immunological memory, which might causelong-lasting regression and prevent relapse in cancer patients.

The composition and characteristics of the tumour microenvironment varywidely and are important in determining the anti-tumour immune response.For example, certain cells of the immune system, including naturalkiller cells, dendritic cells (DCs) and effector T cells, are capable ofdriving potent anti-tumour responses. However, tumour cells often inducean immunosuppressive microenvironment, which favours the development ofimmunosuppressive populations of immune cells, such as myeloid-derivedsuppressor cells and regulatory T cells. Understanding the complexity ofimmunomodulation by tumours is important for the development ofimmunotherapy. Various strategies are being developed to enhanceanti-tumour immune responses, including DC-based vaccines andantagonists of inhibitory signalling pathways to overcome ‘immunecheckpoints’.

The invention provides methods involving administering an anti-TOIligand to a human, the TOI being present in humans as a plurality ofvariants differing by one or more amino acid polymorphisms, the methodcomprising administering a ligand to the human, the ligand comprisingfirst and second protein domains, wherein the first domain specificallybinds a TOI variant comprising a first amino acid polymorphism, whereinthe second domain comprises a second polymorphism, and wherein the humanexpresses (i) TOI comprising said first amino acid polymorphism; and(ii) protein domains comprising said second polymorphism.

Particularly, the invention provides a method of targeting a tumour in ahuman and, eg, in this case the TOI is a tumour antigen (eg, atumour-specific antigen or a tumour-associated antigen, as will be clearto the skilled addressee). For example, the TOI is selected from thegroup consisting of human PD-L1, Alphafetoprotein (AFP),Carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial tumor antigen(ETA) and Melanoma-associated antigen (MAGE).

Optionally the ligand comprises a human cytokine variant amino acidsequence, optionally the cytokine being selected from the groupconsisting of IL-2, IL-12, IL-15 and IL-21, eg, wherein the sequence isfused to the N-terminus of one or more heavy and/or light chains of theligand when the ligand is an antibody or fragment or a trap comprisingFc regions.

In an example, the cytokine is human IL-2 and the sequence comprises apolymorphism selected from 142I, 129D, 76L, 38L and 27T. For example,the sequence comprises 142I and 129D. These positions are indicated tobe polymorphic and associated with SNPs in Ensembl and dbSNP. In anembodiment, the ligand comprises an IL-2 amino acid sequence comprisingone, more or all of the polymorphisms of said group. In an example, thecytokine comprises SEQ ID NO: 134.

In an example, the cytokine is human IL-21 and the sequence comprises apolymorphism selected from 32Q, 40R, 431, 98V, 106K, 112A, 113G, 134Kand 135E. For example, the sequence comprises 32Q and 40R. Thesepositions are indicated to be polymorphic and associated with SNPs inEnsembl and dbSNP. In an embodiment, the ligand comprises an IL-21 aminoacid sequence comprising one, more or all of the polymorphisms of saidgroup.

In an alternative, there is provided a method of cancer immunotherapyand, eg, in this case the TOI is a human immune cell antigen (eg,wherein the antigen is a human natural killer cell, dendritic cell (DC)or effector T cell antigen). In another example the TOI is an immunecheckpoint target.

In an embodiment the TOI is human PD-1. In an embodiment the TOI ishuman PD-L1 or PD-L2. In another embodiment the target is human CTLA-4.In another embodiment the target is human LAG-3. In another embodimentthe target is human TIM-3. In an embodiment the TOI is human OX40 (andoptionally the ligand agonises the TOI). In an embodiment the TOI ishuman KIR. In an embodiment the TOI is human CD137 (and optionally theligand agonises the TOI). In an embodiment the TOI is human CD27 (andoptionally the ligand agonises the TOI). In an embodiment the TOI ishuman CD70. In an embodiment the TOI is human B7-H3. In an embodimentthe TOI is human KIR. In an embodiment the TOI is human GITR (andoptionally the ligand agonises the TOI). In an example, the ligandcomprises a human cytokine variant amino acid sequence as describedabove, the cytokine being useful for immunotherapy of cancer. Where anagonist is not mentioned, the ligand can for example antagonise the TOI.

In an example the ligand is selected from the ligands of Table 22.

TABLE 22 Ligands ipilimumab nivolumab pembrolizumab durvalumabtremelimumab RG-7446 lirilumab MEDI-6469 MSB -0010718C pidilizumabAMP-514 TRX-518 BMS-986016 urelumab varlilumab PF-05082566 SGN-CD70AARGX-110 MGA-271 rHIgM12B7

In an example of either alternative the method treats or reduces therisk of a cancer, eg, any cancer disclosed herein (eg, as disclosed inExample 5, 6 or this Example 7).

Thus, an alternative provides: A method of cancer immunotherapy in ahuman by targeting an immune cell TOI in the human, the TOI beingpresent in humans as a plurality of variants differing by one or moreamino acid polymorphisms, the method comprising administering a ligandto the human, the ligand comprising first and second protein domains,wherein the first domain specifically binds a TOI variant comprising afirst amino acid polymorphism, wherein the second domain comprises asecond polymorphism, and wherein the human expresses (i) TOI comprisingsaid first amino acid polymorphism; and (ii) protein domains comprisingsaid second polymorphism.

The TOI is for example PD-1 (also known as PDCD1, programmed cell death1, PD1 and CD279), CTLA-4, LAG-3 or TIM-3. For example, the TOI is humanPD-1 and the first polymorphism is a PD-1 polymorphism described herein(eg, a varation listed in Table 23). For example the polymorphism isencoded by a PD-1 SNP described herein.

It may be desirable to block (not necessarily clear as with an IgG1format) the TOI in the method of this alternative and a human IgG4 maybe preferable for doing this in a cancer immunotherapy setting. Thus, inan embodiment, the ligand comprises a human gamma-4 constant region,wherein the constant region comprises said second domain. Importantly,according to the invention, the constant region polymorphism is matchedwith the genome of the human, thereby minimising interfering immuneresponses, as may be desirable to minimise in the current cancerimmunotherapy setting. “Blocking” of the TOI means, for example,antagonising the binding of the TOI to a cognate receptor or humanbiological target, eg, blocking PD-1 binding to PD-L1 and/or PD-L2;CTLA-4 binding to B7-1 or B7-2; or CD28 binding to B7-1 or B7-2.

For the cancer immunotherapy setting of the invention, it may beadvantageous wherein the gamma-4 is a gamma-4 constant region with228Pro or 235Glu. In an example the constant region is an IgG4PE (ie, agamma-4 constant region with 228Pro and 235Glu). In an example, theligand comprises human IGHG4*01 hinge S10>P constant region.

In an example, therefore, the ligand comprises any human gamma-4constant region disclosed herein (eg, in any of Examples 1 et seq).

The present invention, thus, provides the following concepts:—

-   -   1. A method of cancer immunotherapy in a human by targeting an        immune cell TOI (eg, PD-1) in the human, the TOI being present        in humans as a plurality of variants differing by one or more        amino acid polymorphisms, the method comprising administering a        ligand (eg, an antibody or antibody fragment) to the human, the        ligand comprising first and second protein domains, wherein the        first domain specifically binds a TOI variant comprising a first        amino acid polymorphism, wherein the second domain comprises a        second polymorphism, and wherein the human expresses (i) TOI        comprising said first amino acid polymorphism; and (ii) protein        domains comprising said second polymorphism, wherein the ligand        comprises a human gamma-4 heavy chain constant region that        comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg        at position 289 shown in SEQ ID NO: 73 and wherein said human        comprises an IGHG4*01 human heavy chain constant region gene        segment, or the human expresses antibodies comprising human        gamma-4 heavy chain constant regions comprising a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73; and wherein the second domain is        comprised by said gamma-4 heavy chain constant region of the        ligand.        -   Optionally, the TOI is a human immune cell antigen (eg,            wherein the antigen is a human natural killer cell,            dendritic cell (DC) or effector T cell antigen).        -   The programmed death-1 (PD-1) receptor serves as an            immunologic checkpoint, limiting bystander tissue damage and            preventing the development of autoimmunity during            inflammatory responses. PD-1 is expressed by activated T            cells and down-modulates T-cell effector functions upon            binding to its ligands, PD-L1 and PD-L2, on            antigen-presenting cells. In patients with cancer, the            expression of PD-1 on tumor-infiltrating lymphocytes and its            interaction with the ligands on tumor and immune cells in            the tumor microenvironment undermines antitumor immunity and            supports the rationale for PD-1 blockade in cancer            immunotherapy. In a particularly preferred embodiment, there            is provided a method of cancer immunotherapy in a human by            targeting PD-1 in the human.    -   2. The method of concept 1, wherein the first and/or second        polymorphism is respectively encoded by a SNP having a        cumulative human allele frequency of less than 50%.        -   Optionally the frequency is less than 45, 40, 35, 30, 25,            20, 15, 10, 5, 4, 3, 2, or 1%.    -   3. The method of any preceding concept, wherein the first domain        is an antibody variable domain or a receptor domain.    -   4. The method of any preceding concept, wherein the second        domain is an antibody domain, optionally an antibody variable or        constant domain.    -   5. The method of any preceding concept, wherein the second        domain is a domain of an antibody Fc region.    -   6. The method of any one of concepts 1 to 4, wherein the first        and second domains are antibody variable domains, optionally        wherein the variable domains specifically bind first and second        human TOI respectively, wherein the TOIs are different.    -   7. The method of any one of concepts 1 to 4, wherein the first        and second domains are receptor domains, optionally wherein the        receptor domains specifically bind first and second human TOI        respectively, wherein the TOIs are different.    -   8. The method of concept 6 or 7, wherein the first domain        comprises a third amino acid polymorphism, wherein the human        expresses (iii) domains comprising said third amino acid        polymorphism.        -   Optionally the human expresses the second TOI variant. In an            example, the second TO is a human immune cell surface            antigen or a tumour antigen.        -   Optionally the third polymorphism is encoded by a SNP having            a cumulative human frequency of less than 50%. Optionally            the frequency is less than 45, 40, 35, 30, 25, 20, 15, 10,            5, 4, 3, 2, or 1%.        -   In an example, the first domain is an antibody variable            domain (eg, any V domain of the invention described herein,            such human VH3-23*04 as herein described) and the domains            of (iii) are antibody variable domains. For example, the            first domains and domains of (iii) are VH domains. For            example, the first domains and domains of (iii) are Vκ            domains. For example, the first domains and domains of (iii)            are Vλ, domains.        -   In an example, the first domain is a receptor binding site            and the domains of (iii) are said receptor binding site            domains.    -   9. A method according to concepts 1, 2 and 8 combined.    -   10. The method of any one of concepts 1 to 3, wherein the second        domain is a domain of a human cytokine variant, optionally        selected from the group consisting of IL-2, IL-12, IL-15 and        IL-21, and the domains of (ii) are domains of said cytokine        variant.        -   In an example, the second domain is a domain of human IL-2            and the second polymorphism is selected from the group            consisting of 1421, 129D, 76L, 38L and 27T. For example, the            second domain comprises 1421 and 129D. These positions are            indicated to be polymorphic and associated with SNPs in            Ensembl and dbSNP. In an embodiment, the ligand comprises an            IL-2 amino acid sequence comprising one, more or all of the            polymorphisms of said group.        -   In an example, the second domain is a domain of human IL-21            and the second polymorphism is selected from the group            consisting of 32Q, 40R, 431, 98V, 106K, 112A, 113G, 134K and            135E. For example, the second domain comprises 32Q and 40R.            These positions are indicated to be polymorphic and            associated with SNPs in Ensembl and dbSNP. In an embodiment,            the ligand comprises an IL-21 amino acid sequence comprising            one, more or all of the polymorphisms of said group.    -   11. The method of any preceding concept, wherein the human        expresses the first and/or second domain.    -   12. The method of any preceding concept, wherein the first,        second and/or third polymorphism is respectively found in at        least 5 different human populations as defined in Table 4.        -   For example, each of the first and second polymorphisms is            respectively found in at least 5 different human populations            as defined in Table 4. For example, each of the first,            second and polymorphisms is respectively found in at least 5            different human populations as defined in Table 4.        -   Optionally the first, second and/or third (see below)            polymorphism respectively appears in at least 9, 10, 11, 12,            13, 14, 15, 16, 17, 18, 19 or 20 different human populations            as defined in Table 4.    -   13. The method of concept 12, wherein the first, second and/or        third polymorphism respectively appears in at least 15 humans in        the 1000 Genomes Phase I database.    -   In an embodiment, the first, second and/or third polymorphism is        found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,        75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150        human individuals distributed across such many different human        populations.    -   14. The method of any preceding concept, wherein the first,        second and/or third polymorphism is respectively found in at        least 5 different human populations as defined in Table 4 and is        encoded by a SNP having a cumulative human allele frequency of        less than 50%.        -   Optionally the frequency is less than 45, 40, 35, 30, 25,            20, 15, 10, 5, 4, 3, 2, or 1%.    -   15. The method of any preceding concept, wherein the first,        second and/or third polymorphism is respectively encoded by a        SNP having total human genotype frequency of less than 50%.        -   Optionally the frequency is less than 45, 40, 35, 30, 25,            20, 15, 10, 5, 4, 3, 2, or 1%.    -   16. The method of any preceding concept, wherein the first and        second domains are human, optionally wherein the ligand is fully        human.    -   17. The method of any preceding concept, wherein the ligand is        an anti-TOI trap, antibody or antibody fragment.    -   18. The method of any preceding concept, wherein the ligand        specifically binds two or more human TOIs, eg, said first and        second TOIs.    -   19. The method of any preceding concept, wherein the ligand is        administered to the human by injection or the ligand is provided        by an injectable preparation.    -   20. The method of any preceding concept, wherein said human has        been or is genotyped as positive for said TOI variant nucleotide        sequence before administering the ligand, or the method        comprises genotyping the human as positive for said variant        nucleotide sequence before administering the ligand.    -   21. The method of any preceding concept, wherein the human has        been or is phenotyped as positive for said TOI variant before        administering the ligand, or the method comprises phenotyping        the human as positive for said variant before administering the        ligand.    -   22. The method of any preceding concept, comprising, before said        administering, selecting a human comprising said first TOI        polymorphism, wherein the human is the human of concept 1.    -   23. The method of any preceding concept, wherein the human has        been determined to comprise the first and/or second TOI variant        or a nucleotide sequence encoding the TOI variant(s).    -   24. The method of any preceding concept, comprising the step of        determining that the human comprises a first TOI nucleotide        sequence encoding the first polymorphism, optionally, wherein        the determining step is performed before administration of the        ligand to the human.    -   25. The method of concept 24 wherein the step of determining        comprises assaying a biological sample from the human for said        nucleotide sequence.    -   26. The method of concept 25, wherein the assaying comprises        contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence comprising a SNP encoding the first            polymorphism or that specifically hybridizes to an antisense            of said sequence, wherein said nucleic acid hybridizes to            said SNP or hybridizes to an antisense sequence thereby            forming a complex when at least one nucleotide sequence            comprising said SNP is present; and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence comprising said SNP or comprising an antisense            sequence of said contiguous nucleotides, wherein said            sequence of contiguous nucleotides comprises said SNP            thereby forming a complex when the nucleotide sequence            comprising said SNP is present; and        -   c. detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises the first TOI nucleotide sequence.    -   27. The method of concept 25, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   28. The method of any preceding concept, wherein said human is        or has been further determined to be substantially resistant to        a cancer treatment.    -   29. The method of any preceding concept, wherein said human is        receiving or has received cancer treatment or has reduced        responsiveness to a cancer treatment.    -   30. The method of concept 25, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.    -   31. The method of any preceding concept, wherein said human has        been diagnosed with said disease or condition.    -   32. An anti-TOI ligand (eg, an antibody, antibody fragment or        trap) for use in the method of any preceding concept. For        example, the TOI is human PD-1 (eg, the ligand is is        pembrolizumab, lambrolizumab or KEYTRUDA™. In an example, the        ligand is nivolumab or OPDIVO™) For example, the TOI is human        CTLA-4, eg, the ligand is ipilimumab, YERVOY™ tremelimumab,        ticilimumab, belatacept or NulojiX™.        -   In an embodiment, the trap is a receptor Fc fusion (ie, a            receptor domain fused to an antibody Fc region, eg, as a            dimer), wherein the receptor specifically binds to the TOI.    -   33. The ligand of concept 32, wherein the ligand is fully human.    -   34. The ligand of concept 32 or 33, wherein the ligand is for        administration by injection or the ligand is provided by an        injectable preparation; wherein the ligand is for topical or        inhaled administration or the ligand is provided by a topical or        inhalable preparation.

In an example, the first, second and/or third polymorphismrespectively:—

-   -   d. has a cumulative human allele frequency of 15% or less;    -   e. has a total human genotype frequency of 20% or less;    -   f. is found in at least 5 different human populations (using the        standard categorisation of the 1000 Genomes Project, eg, as per        the Phase I database); and    -   g. is found in many individuals distributed across such many        different populations.    -   In an example, the criteria are applied with reference to one or        more human genomic sequence databases as described herein. For        example, the criteria are those as applied to the 1000 Genomes        database.    -   For example in any aspect example, embodiment or configuration        of the invention, the 1000 Genomes database release 13 or        Phase I. For example, the 1000 Genomes database in its most        recent version as at 1 Oct. 2013 or 1 Oct. 2014.    -   In an example, the human is homozygous for the first        polymorphism. Additionally or alternatively, the human is        homozygous for the first polymorphism. Additionally or        alternatively, the human is homozygous for the third        polymorphism. For example, the human is homozygous for the first        and second polymorphisms.    -   In an example, the method is according to any one of the        disclosure herein involving selecting, genotyping or phenotyping        the human as positive for the TOI variant (ie, in the present        case human PD-1 comprising the polymorphism or a nucleotide        sequence encoding this). In an example, the method is according        to any one of concepts above involving the determining step or        wherein the human has been determined to comprise the variant        TOI variant (eg, in the present case human PD-1 comprising the        polymorphism or a nucleotide sequence encoding this).    -   In an example, the first polymorphism is a human PD-1 amino acid        polymorphism described herein or is encoded by a human PD-1 SNP        described herein.    -   In an example, wherein the TOI is human PD-1 the first        polymorphism is encoded by a SNP selected from the group        consisting of rs7568402, rs28699177, rs28542728, rs118117097,        rs28570544, rs118027315, rs191919871, rs6707556 and rs142909968.

The invention further provides the following specific aspects:—

-   -   1. A method of cancer immunotherapy in a human by targeting PD-1        in the human, the method comprising administering an antibody or        antibody fragment to the human, wherein the antibody or antibody        fragment specifically binds a PD-1 comprising a first amino acid        polymorphism, wherein the antibody or antibody fragment        comprises a human gamma-4 heavy chain constant region that        comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg        at position 289 shown in SEQ ID NO: 73 and wherein said human        comprises (i) an IGHG4*01 human heavy chain constant region gene        segment, or the human expresses antibodies comprising human        gamma-4 heavy chain constant regions comprising a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence        encoding a PD-1 comprising said first amino acid polymorphism or        the human expresses a PD-1 comprising said first amino acid        polymorphism.    -   A first alternative aspect 1 provides:—    -   A method of cancer immunotherapy in a human by targeting PD-1 in        the human, the method comprising administering an antibody or        antibody fragment to the human, wherein the antibody or antibody        fragment specifically binds a PD-1 encoded by a PD-1 nucleotide        sequence comprising a SNP (first polymorphism) selected from the        group consisting of the variations set out in Table 23 (eg,        selected from the group consisting of rs36084323, rs10204225,        rs11568821, rs2227981 and rs2227982), wherein the antibody or        antibody fragment comprises a human gamma-4 heavy chain constant        region that comprises a Leu at position 189 shown in SEQ ID NO:        73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein        said human comprises (i) an IGHG4*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-4 heavy chain constant regions comprising        a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at        position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide        sequence comprising said selected SNP.    -   These SNPs are discussed further below.    -   A first alternative aspect 1 provides:—    -   A method of treating cancer in a human, the method comprising        administering an antibody or antibody fragment to the human,        wherein the antibody or antibody fragment inhibits the binding        of PD-L1 or PD-L2 to a PD-1 comprising a first amino acid        polymorphism, wherein the antibody or antibody fragment        comprises a human gamma-4 heavy chain constant region that        comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg        at position 289 shown in SEQ ID NO: 73 and wherein said human        comprises (i) an IGHG4*01 human heavy chain constant region gene        segment, or the human expresses antibodies comprising human        gamma-4 heavy chain constant regions comprising a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide sequence        encoding a PD-1 comprising said first amino acid polymorphism or        the human expresses a PD-1 comprising said first amino acid        polymorphism. In an example, the human comprises a PD-1        nucleotide sequence comprising a SNP selected from the group        consisting of a PD-L1 SNP disclosed in Example 6, or PD-1 SNP        rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982;        optionally wherein the human has been determined to comprise        said SNP or the method comprises determining that the human        comprises said SNP before administering the antibody or        fragment.

In an example the antibody or fragment specifically binds human PD-L1,PD-L2 or PD-1. In an example the antibody or fragment specifically bindshuman PD-L1. In an example the antibody or fragment specifically bindshuman PD-1.

For example, the PD-L1 comprises a PD-L1 polymorphism described inExample 6 or is encoded by a PD-L1 nucleotide sequence comprising aPD-L1 SNP described in Example 6.

For example, the PD-1 comprises a PD-1 polymorphism described herein(eg, in Table 23) or is encoded by a PD-1 nucleotide sequence comprisinga PD-1 SNP described herein (eg, in Table 23), eg, a SNP selected fromthe group consisting of rs36084323, rs10204225, rs11568821, rs2227981and rs2227982. These SNPs are discussed further below.

-   -   2. The method of aspect 1, wherein the first polymorphism or        selected SNP is a nucleotide corresponding to SNP rs36084323,        rs10204225, rs11568821, rs2227981 or rs2227982.    -   3. The method of aspect 1 or 2, wherein said human is homozygous        for said first polymorphism or selected SNP.    -   4. The method of any preceding aspect, wherein the ligand        comprises an IGHG4*01 human heavy chain constant region.    -   5. The method of any preceding aspect, wherein the cancer is        selected from the group consisting of renal cell carcinoma;        haematological neoplasm; stage IV melanoma; non small-cell lung        cancer; metastatic stomach cancer; breast cancer; solid tumour;        bladder cancer; head and neck cancer, colorectal cancer,        glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's        lymphoma, follicular lymphoma, cervical cancer, myeloid        leukaemia (eg, acute myeloid leukaemia or chronic myeloid        leukaemia). In an embodiment, the cancer is not melanoma. In an        embodiment, the cancer is not lung cancer. In an embodiment, the        cancer is not non small-cell lung cancer.    -   6. The method of any preceding aspect, comprising, before said        administering, selecting a human comprising said PD-1 nucleotide        sequence of (ii), wherein the human is the human of aspect 1.    -   7. The method of any preceding aspect, wherein the human has        been determined to comprise said PD-L nucleotide sequence of        (ii).    -   8. The method of any preceding aspect, comprising the step of        determining that the human comprises said PD-L nucleotide        sequence of (ii), optionally, wherein the determining step is        performed before administration of the antibody to the human.    -   9. The method of aspect 8, wherein the step of determining        comprises assaying a biological sample from the human for a PD-L        nucleotide sequence comprising said selected variation.    -   10. The method of aspect 9, wherein the assaying comprises        contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence comprising said selected variation or            that specifically hybridizes to an antisense of said            sequence, wherein said nucleic acid hybridizes to said            selected variation or hybridizes to an antisense sequence            thereby forming a complex when at least one nucleotide            sequence comprising said selected variation is present;            and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence comprising said selected variation or comprising an            antisense sequence of said contiguous nucleotides, wherein            said sequence of contiguous nucleotides comprises said            selected variation thereby forming a complex when the            nucleotide sequence comprising said selected variation is            present; and        -   c. detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises a PD-L1 nucleotide sequence comprising said            selected variation.    -   11. The method of aspect 10, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   12. The method of any preceding aspect, wherein said human is or        has been further determined to be substantially resistant a        cancer, PD-L1 or PD-1 treatment.        -   For example the human is or has been further determined to            be resistant to a chemotherapy treatment.    -   13. The method of any preceding aspect, wherein said human is        receiving or has received a cancer (eg, chemotherapy), PD-L1 or        PD-1 treatment or has reduced responsiveness to a cancer (eg,        chemotherapy), PD-L1 or PD-1 treatment.    -   14. The method of aspect 9, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.    -   15. The method of any preceding aspect, wherein said ligand is        administered by intravenous or subcutaneous injection and/or is        comprised in an injectable preparation.    -   16. The method of any preceding aspect, wherein the ligand is        human, eg, a human antibody or antibody fragment.    -   17. An anti-human PD-1 ligand (eg, an antibody, antibody        fragment or trap) for use in the method of any preceding aspect.        -   For example, the ligand is is pembrolizumab, lambrolizumab            or KEYTRUDA™. In an example, the ligand is nivolumab or            OPDIVO™)        -   In an embodiment, the trap is a receptor Fc fusion (ie, a            receptor domain fused to an antibody Fc region, eg, as a            dimer), wherein the receptor specifically binds to human            PD-1.    -   18. The ligand of aspect 17, wherein the ligand is fully human.    -   19. The ligand of aspect 17 or 18, wherein the ligand is for        administration by injection or the ligand is provided by an        injectable preparation; wherein the ligand is for topical or        inhaled administration or the ligand is provided by a topical or        inhalable preparation.

Optionally in any of the configurations, concepts, aspects, embodimentsor examples, one or more of the following optional features applies.

Optionally, the cancer is selected from the group consisting of renalcell carcinoma; haematological neoplasm; stage IV melanoma; nonsmall-cell lung cancer; metastatic stomach cancer; breast cancer; solidtumour; bladder cancer; head and neck cancer, colorectal cancer,glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin'slymphoma, follicular lymphoma, cervical cancer, myeloid leukaemia (eg,acute myeloid leukaemia or chronic myeloid leukaemia). In an embodiment,the cancer is not melanoma. In an embodiment, the cancer is not lungcancer. In an embodiment, the cancer is not non small-cell lung cancer.

Optionally, the cancer is renal cell cancer.

Optionally, the cancer is locally advanced or metastatic carcinoma,locally advanced or metastatic melanoma, or locally advanced ormetastatic non-small cell lung carcinoma.

Optionally, the cancer is urothelial cancer.

Optionally, the cancer is recurrent or metastatic head and neck squamouscell carcinoma.

Optionally, the cancer is PD-L1-positive advanced or metastaticnon-small cell lung cancer.

Optionally, the cancer is multiple myeloma.

Optionally, the cancer is advanced melanoma.

Optionally, the cancer is advanced Merkel cell cancer.

Optionally, the cancer is solid tumour, eg, advanced solid tumours.

In an example, the ligand comprises a human IgG4PE constant region.

In an example, the ligand is pembrolizumab, lambrolizumab or KEYTRUDA™.In an example, the ligand is nivolumab or OPDIVO™

The TOI polymorphism is associated, for example, with a cancer, such asany cancer disclosed herein.

The invention also provides an anti-TOI ligand (eg, a human PD-1, PD-L1or PD-L2 trap, or anti-human PD-1, PD-L1 or PD-L2 antibody or fragment)for use in any cancer treatment or immunotherapy method of theinvention.

Optionally, the ligand (eg, an antibody or antibody fragment) isaccording to one of the following options:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment selected from the group consisting ofIGHV3-33*01 and IGHV3-7*01, a human D gene segment and a human JHsegment, and wherein said human comprises said selected VH gene segmentor the human expresses VH domains derived from the recombination of saidselected human VH segment, a human D gene segment and a human JHsegment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment selected from the group consisting ofIGKV3-11*01 and IGKV1-12*01 and a human Jκ segment, and wherein saidhuman comprises said selected Vκ gene segment or the human expresses Vκdomains derived from the recombination of said selected human Vκ segmentand a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.

Optionally, instead of a gamma-4 constant region, the ligand (eg, atrap, antibody or fragment) comprises a heavy chain constant regionaccording to any of the following options:—(v) wherein the ligandcomprises a human gamma-1 heavy chain constant region that comprises anAsp at position 204 shown in SEQ ID NO: 4 or a Leu at position 206 shownin SEQ ID NO: 4 and wherein said human comprises (i) an IGHG1*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-1 heavy chain constant regionscomprising an Asp at position 204 shown in SEQ ID NO: 4 or a Leu atposition 206 shown in SEQ ID NO: 4. (vi) wherein the ligand comprises ahuman gamma-2 heavy chain constant region that comprises an amino acidselected from the group consisting of a Pro at position 72 shown in SEQID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID NO: 6and an Ala at position 257 shown in SEQ ID NO: 6 and wherein said humancomprises (i) an IGHG2*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-2heavy chain constant regions comprising said selected Pro at position 72shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe atposition 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ IDNO: 6 or Ala at position 257 shown in SEQ ID NO: 6. In an example, theconstant region is Fc enhanced, eg, ADCC or CDC enhanced. The skilledperson will know techniques (eg, known C region mutations) to enhanceADCC or CDC.

(vii) wherein the ligand comprises a human gamma-3 heavy chain constantregion encoded by a first human IGHG3 (eg, IGHG3*01) constant regiongene segment and wherein said human comprises (i) said first constantregion gene segment (eg, an IGHG3*01), or the human expresses antibodiescomprising human gamma-3 heavy chain constant regions encoded by saidfirst human IGHG3 (eg, IGHG3*01) constant region gene segment.

Optionally, the ligand (eg, an antibody or antibody fragment) isaccording to one of the following options:—

(viii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(ix) wherein the ligand comprises a human IGLC1*01 lambda chain constantregion and wherein said human comprises (i) a human IGLC1*01 lambdachain constant region gene segment, or the human expresses antibodiescomprising human IGLC1*01 lambda chain constant regions.

-   -   (x) wherein the ligand comprises a human epsilon heavy chain        constant region encoded by a first human epsilon heavy chain        constant region gene segment and wherein said human        comprises (i) said first constant region gene segment, or the        human expresses antibodies comprising human epsilon heavy chain        constant regions encoded by said first constant region gene        segment.    -   (xii) wherein the ligand comprises a human mu heavy chain        constant region encoded by a first human mu heavy chain constant        region gene segment and wherein said human comprises (i) said        first constant region gene segment, or the human expresses        antibodies comprising human mu heavy chain constant regions        encoded by said first constant region gene segment.    -   (xiii) wherein the ligand comprises a human alpha heavy chain        constant region encoded by a first human alpha heavy chain        constant region gene segment and wherein said human        comprises (i) said first constant region gene segment, or the        human expresses antibodies comprising human alpha heavy chain        constant regions encoded by said first constant region gene        segment.    -   (xiv) wherein the ligand comprises a human delta heavy chain        constant region encoded by a first human delta heavy chain        constant region gene segment and wherein said human        comprises (i) said first constant region gene segment, or the        human expresses antibodies comprising human delta heavy chain        constant regions encoded by said first constant region gene        segment.    -   (xv) wherein the ligand comprises a human kappa light chain        constant region encoded by a first human kappa light chain        constant region gene segment and wherein said human        comprises (i) said first constant region gene segment, or the        human expresses antibodies comprising human kappa light chain        constant regions encoded by said first constant region gene        segment.    -   (xvi) wherein the ligand comprises a human lambda light chain        constant region encoded by a first human lambda light chain        constant region gene segment and wherein said human        comprises (i) said first constant region gene segment, or the        human expresses antibodies comprising human lambda light chain        constant regions encoded by said first constant region gene        segment.

References

-   (a) Cancer Immunol Res. 2014 September; 2(9):846-56. doi:    10.1158/2326-6066.CIR-14-0040. Epub 2014 May 28; “In vitro    characterization of the anti-PD-1 antibody nivolumab, BMS-936558,    and in vivo toxicology in non-human primates”, Wang C et al.-   (b) Clin Biochem. 2014 May; 47(7-8):612-7. doi:    10.1016/j.clinbiochem.2013.12.023. Epub 2014 Jan. 2; “Programmed    death-1 (PD-1) polymorphisms in Chinese patients with esophageal    cancer”; Qiu H et al.-   (c) Gene. 2012 Oct. 25; 508(2):229-32. doi:    10.1016/j.gene.2012.07.059. Epub 2012 Aug. 8; “Programmed death-1    gene polymorphism (PD-1.5 C/T) is associated with colon cancer”;    Mojtahedi Z et al.-   (d) Gastroenterol Hepatol Bed Bench. 2013 Fall; 6(4):178-82;    “Programmed death-1 gene polymorphism (PD-1.5 C/T) is associated    with gastric cancer”; Savabkar S et al.-   (e) Breast Cancer Res Treat. 2011 August; 129(1):195-201. doi:    10.1007/s10549-011-1440-3. Epub 2011 Apr. 13; “PD-1 polymorphisms    are associated with sporadic breast cancer in Chinese Han population    of Northeast China”; Hua Z et al.

Human Polymorphisms & SNPs

The inventor carried out assessment of natural human variation in PD-1and identified the following variations of interest for application tothe present invention.

TABLE 23 Human PD-1 Variation CHROMOSOMAL LOCATION AMINO AMINO SNP/(FORWARD NUCLEOTIDE ACID ACID CUMULATIVE VARIANT STRAND) CHANGE¹ CHANGE²POSITION³ FREQUENCY⁴ rs7568402 rs28699177 rs28542728 rs118117097rs28570544 rs118027315 rs191919871 rs6707556 rs142909968 rs36084323⁺ 2:241859444 C > T ^(0.17) rs10204225 2: 197610964 G > A rs11568821⁺⁺ 2:241851760 C > T ^(0.05) rs2227981⁺⁺⁺ 2: 241851121 G > A ^(0.38)rs2227982⁺⁺⁺⁺ 2: 241851281 G > A A215V 215 ^(0.160) ^(1,2,3,4)Accordingto Ensembl, dbSNP; with reference to transcript ENST00000334409 (thesequence of this transcript being incorporated herein in its entirety).⁺rs36084323 is present in humans at an average cumulative frequency of17% according to the 1000 Genomes Phase I database, but is higher inAMR, ASN, MXL, CHB, CHS and JPT populations; thus in an embodiment, thehuman is of ASN ancestry, eg, of MXL, CHB, CHS or JPT ancestry.⁺⁺rs11568821 is present in humans at an average cumulative frequency of5% according to the 1000 Genomes Phase I database, but is higher in AMR,EUR, PUR, CEU, GBR, IBS and TSI populations; thus in an embodiment, thehuman is of AMR or EUR ancestry, eg, of CEU, GBR, IBS or TSI ancestry.⁺⁺⁺rs2227981 is present in humans at an average cumulative frequency of38% according to the 1000 Genomes Phase I database, but is higher inAFR, EUR, ASW, LWK, YRI, CEU, GBR, IBS and TSI populations; thus in anembodiment, the human is of AFR or EUR ancestry, eg, of ASW, LWK, YRI,CEU, GBR, IBS or TSI ancestry. ⁺⁺⁺rs2227982 is present in humans at anaverage cumulative frequency of 16% according to the 1000 Genomes PhaseI database, but is higher in ASN, CHB, CHS and JPT populations; thus inan embodiment, the human is of ASN ancestry, eg, of CHB, CHS or JPTancestry.

Using this analysis, the inventor has devised the configurations,aspects, concepts, examples and embodiments of the invention set out inthis Example 7 that are useful for addressing diseases and conditions,such as cancer, autoimmune or inflammatory diseases and conditions.

In an embodiment, the human comprises a SNP selected from the SNPs incolumn 1 of Table 23; optionally also the ligand specifically binds aPD-1 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the ligand (eg, trap, antibody or antibody fragment)specifically binds a PD-1 comprising a corresponding variation as shownin Table 23 or is encoded by a nucleotide sequence comprising orencoding a SNP or amino acid variation shown in Table 23.

For example the SNP is selected from rs36084323, rs10204225, rs11568821,rs2227981 and rs2227982. For example the SNP is rs36084323. For examplethe SNP is rs10204225. For example the SNP is rs11568821. For examplethe SNP is rs2227981. For example the SNP is rs2227982.

In an example the SNP and indication are a SNP/indication combinationmentioned in any of the references disclosed in this Example 7.

In an example, the indication is aplastic anaemia and the SNP is a G atthe position of rs36084323.

Example 7B Autoimmune, Inflammatory & Other Disease Applications

In an alternative, instead of “A method of cancer immunotherapy” or “Amethod of treating cancer”, the invention as expressed in Example 7A caninstead be read as “A method of treating or reducing the risk of anautoimmune disease or condition” or “A method of treating or reducingthe risk inflammatory disease or condition” and the TOI polymorphism isassociated with an autoimmune or inflammatory disease or condition. Theother non-cancer features of this example apply mutatis mutandis tothese alternatives. The TOI (eg, PD-1) comprises said first polymorphismor is encoded by a nucleotide sequence comprising said selected SNP.

For example, the autoimmune disease or condition is selected from thegroup consisting of rheumatoid arthritis, diabetes (eg, type I diabetesmellitus), Grave's disease, multiple sclerosis and systemic lupuserythematosus.

For example, the inflammatory disease or condition is selected from thegroup consisting of rheumatoid arthritis, ankylosing spondylitis,psoriasis, asthma, colitis and atopic dermatitis. For example, themethod is for treating or reducing the risk of ankylosing spondylitis.In an example, the human is of Chinese (eg, CHS) ancestry.

References

-   (a) Vibanco-Pérez N, Gutiérrez-Franco J, Duran-Avelar M,    Agraz-Cibrian J, Jiménez-Alvarez M, Peña-Virgen S and    Zambrano-Zaragoza J (2013). Association of PD1.1 and PD1.6    polymorphisms with ankylosing spondylitis. Front. Immunol.    Conference Abstract: 15th International Congress ofImmunology (ICI).    doi: 10.3389/conf.fimmu.2013.02.0079. Various SNPs in the PD1 gene    have been identified, such as PD1.1 (rs36084323), PD1.3    (rs11568821), PD1.5 (rs2227981), and PD1.9 (rs2227982). Among these,    PD1.3, PD1.5, and PD1.9 have been associated with autoimmune    disorders in different human populations. PD1.1 and PD1.6 are two    single nucleotide polymorphisms, at −600 G/A (rs36084323), and +8669    G/A (rs10204225) respectively, that have been associated with others    autoimmune diseases such as rheumatoid arthritis, type I diabetes    mellitus, and systemic lupus erythematosus.-   (b) Rheumatol Int. 2011 February; 31(2):209-13. doi:    10.1007/s00296-009-1264-1. Epub 2009 Dec. 12; “Programmed cell death    1 gene polymorphisms is associated with ankylosing spondylitis in    Chinese Han population”; Liu X et al.    -   The genotype distributions of PD-1.9 were reportedly        significantly different between the patients with ankylosing        spondylitis and the controls (P=0.025). The frequencies of TC        genotype and T allele of PD-1.9 were higher in the patients than        those in the controls (P=0.026 and 0.004). Moreover, the        frequency of the CT haplotype (PD-1.5 C/T, PD-1.9 T/C) was        significantly higher in AS patients than the controls (21.6 vs.        13.9%, P=0.002). The CC haplotype was more common in the        controls than in the patients.-   (c) Tissue Antigens. 2003 December; 62(6):492-7; “Association of a    putative regulatory polymorphism in the PD-1 gene with    susceptibility to type 1 diabetes”, Nielsen C et al.    -   Human PD-1 intronic 7146G/A SNP was found to be associated with        the development of type 1 diabetes in humans.-   (d) Inflammation. 2011 December; 34(6):707-12. doi:    10.1007/s10753-010-9282-4; “Study of programmed cell death 1 (PDCD1)    gene polymorphims in Iranian patients with ankylosing spondylitis”,    Soleimanifar N et al.-   (e) Clin Exp Rheumatol. 2011 Janurary-February; 29(1):13-8. Epub    2011 Feb. 23; “Association of polymorphisms in the programmed cell    death 1 (PD-1) and PD-1 ligand genes with ankylosing spondylitis in    a Chinese population”; Yang Q et al.-   (f) Arthritis Rheum. 2005 June; 52(6):1665-9; “Association of a    PDCD1 polymorphism with renal manifestations in systemic lupus    erythematosus”; Johansson M et al.-   (g) Arthritis Rheum. 2005 April; 52(4):1058-62; “A new haplotype of    PDCD1 is associated with rheumatoid arthritis in Hong Kong Chinese”;    Kong E K et al.-   (h) Ann Neurol. 2005 July; 58(1):50-7; “A PD-1 polymorphism is    associated with disease progression in multiple sclerosis”; Kroner A    et al.-   (i) Leuk Lymphoma. 2013 October; 54(10):2251-4. doi:    10.3109/10428194.2013.772605. Epub 2013 Mar. 4; “Association between    polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han    population; Wu Z et al.

Example 8 Iron Regulation

The existence of multiple inherited disorders of iron metabolismsuggests genetic contributions to iron deficiency. To this end, theinventor assessed naturally-occurring variation in human proteins of theiron homeostasis pathway, and for example thehemojuvelin-BMP6-hepcidin-ferroportin axis.

BMP6 is also known as bone morphogenetic protein 6, BMP-6, VGR-1,VG-1-R, VG-1-related protein, Vgl-related sequence, vegetal-related(TGFB related) cytokine and vegetal related growth factor(TGFB-related).

Matriptase-2 (also known as TMPRSS6) is a critical regulator of theiron-regulatory hormone hepcidin in the liver; matriptase-2 cleavesmembrane-bound hemojuvelin and consequently alters bone morphogeneticprotein (BMP) signaling. Hemojuvelin and hepcidin play a critical rolein retinal iron homeostasis; they are also expressed in the retina.

The invention provides methods of iron homeostasis involvingadministering anti-TOI ligand to a human, the TOI being present inhumans as a plurality of variants differing by one or more amino acidpolymorphisms, the method comprising administering a ligand to thehuman, the ligand comprising first and second protein domains, whereinthe first domain specifically binds a TOI variant comprising a firstamino acid polymorphism, wherein the second domain comprises a secondpolymorphism, and wherein the human expresses (i) TOI comprising saidfirst amino acid polymorphism; and (ii) protein domains comprising saidsecond polymorphism. Particularly useful are methods wherein the TOI isselected from the humanhemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.

A: Indications & Humans

Optionally the method is a method for increasing or maintainingincreased blood iron, eg, for treating or reducing the risk of anaemia;or treating iron deficiency; or for treating or reducing the risk ofAnaemia of Chronic Inflammation (ACI); or for treating or reducing therisk of Anaemia of Chronic Disease (ACD); or for treating or reducingthe risk of anaemia associated with cancer, a kidney condition or GvHD;or for treating or reducing the risk of hemochromatosis.

In an embodiment, the patient is a haemodialysis patient and optionallyTOI is selected from the humanhemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis (eg, theTOI is TMPRSS6 that comprises the A736V polymorphism; or eg, the humancomprises the A736V polymorphism).

In an embodiment, the patient is a coronary heart disease patient. In anembodiment, the human is suffering from an inflammatory disease orcondition, eg, an acute acute inflammation.

In an embodiment, the method is for treating or reducing the risk ofhaemochromatosis.

In an embodiment, the method is for reducing or maintaining reducedhepcidin level in the human.

In an embodiment, the method is for regulating (eg, increasing; eg,decreasing) erythropoiesis in the human and optionally TOI is selectedfrom the human hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrinaxis (eg, the TOI is TMPRSS6 that comprises the A736V polymorphism; oreg, the human comprises the A736V polymorphism).

In an embodiment, the method is for treating or reducing the risk ofiron overload in the human.

In an embodiment, the method is for treating or reducing the risk ofiron-refractory iron deficiency anemia (IRIDA) in the human.

B: Human Polymorphisms & SNPs

Any feature of this section B is combinable with any feature of sectionA above. For example, the human and/or TOI comprises a SNP as describedbelow and the method or ligand of the invention is according to one ormore features of A, comprising administering an anti-TOI ligand.

The inventor carried out assessment of natural human variation in genesand proteins involved in iron homeostasis in humans and identified thefollowing variations of interest for application to the presentinvention.

TABLE 24 Iron Homeostasis - Human Polymorphism & SNPs CHROMOSOMALLOCATION AMINO AMINO SNP/ (FORWARD NUCLEOTIDE ACID ACID CUMULATIVEVARIANT STRAND) CHANGE¹ CHANGE² POSITION³ FREQUENCY⁴ BMP6⁵ rs111588693* 6: 7727038 G > A R28Q 28 0.338 TRANSFERRIN rs3811647**  3: 133765185G > A 0.34 TMPRSS6⁶ Y141C I212T R271Q S304L C510S rs855791*** 22:37066896 A > G A736V 736 0.398 rs1421312**** 22: 37091770 A > Grs2543519 22: 37084229 A > G I430T 0.245 rs2235324 22: 37089684 T > CK244W 244 0.393 HFE⁷ rs1799945  6: 26090951 C > G H63D 63 0.084rs1800562  6: 26092913 G > A C282Y 282 0.020 CUBN⁸ rs10904850# 10:16955708 G > A 0.21 rs1801224 10: 17105522 T > G P389T 389 0.440FERROPORTIN⁹ rs11568350##  2: 189565370 C > A Q248H 248 0.010HAEMOJUVELIN¹⁰ rs7540883###  1: 146018429 C > G A310G 310 0.022HEPCIDIN¹¹ rs146776859 19: 35284790 C > T T31M 31 0.002 MISCELLANEOUSrs987710 22: 22158022 G > A 0.38 rs2698530  2: 64276761 A > C 0.33^(1,2,3,4)According to Ensembl, dbSNP; ⁵with reference to transcriptENST00000283147 (the sequence of this transcript being incorporatedherein in its entirety). ⁶with reference to transcript ENST00000346753(the sequence of this transcript being incorporated herein in itsentirety); with the exception of rs2543519, which is with reference totranscript ENST00000442782 (the sequence of this transcript beingincorporated herein in its entirety); ⁷with reference to transcriptENST00000309234 (the sequence of this transcript being incorporatedherein in its entirety); ⁸with reference to transcript ENST00000377833(the sequence of this transcript being incorporated herein in itsentirety); ⁹with reference to transcript ENST00000261024 (the sequenceof this transcript being incorporated herein in its entirety); ¹⁰withreference to transcript ENST00000336751 (the sequence of this transcriptbeing incorporated herein in its entirety); ¹¹with reference totranscript ENST00000222304 (the sequence of this transcript beingincorporated herein in its entirety); *rs111588693 is present in humansat an average cumulative frequency of 34% according to the 1000 GenomesPhase I database, but is higher in EUR, CEU, FIN, GBR, IBS and TSIpopulations; thus in an embodiment, wherein the method involves BMP6, egadministering an anti-BMP6 ligand, the human is of EUR ancestry, eg, ofCEU, FIN, GBR, IBS or TSI ancestry. **rs3811647 is present in humans atan average cumulative frequency of 34% according to the 1000 GenomesPhase I database, but is higher in ASN, CHB, CHS and JPT populations;thus in an embodiment, wherein the method involves transferrin, egadministering an anti-transferrin ligand, the human is of ASN ancestry,eg, of CHB, CHS or JPT ancestry. ***rs855791 is present in humans at anaverage cumulative frequency of 40% according to the 1000 Genomes PhaseI database, but is higher in AMR, ASN, CLM, MXL, PUR, CHB, CHS and JPTpopulations; thus in an embodiment, wherein the method involves TMPRSS6,eg administering an anti-TMPRSS6 ligand, the human is of AMR or ASNancestry, eg, of CLM, MXL, PUR, CHB, CHS or JPT ancestry. ****rs1421312is present in humans at an average cumulative frequency of 44% accordingto the 1000 Genomes Phase I database, but is higher in AFR, ASW, LWK andYRI populations; thus in an embodiment, wherein the method involvesTMPRSS6, eg administering an anti-TMPRSS6 ligand, the human is of AFRancestry, eg, of ASW, LWK or YRI ancestry. #rs10904850 is present inhumans at an average cumulative frequency of 21% according to the 1000Genomes Phase I database, but is higher in EUR, CEU, FIN, GBR, IBS andTSI populations; thus in an embodiment, wherein the method involvesCUBN, eg administering an anti-CUBN ligand, the human is of EURancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry. ##rs11568350 ispresent in humans at an average cumulative frequency of 1% according tothe 1000 Genomes Phase I database, but is higher in AFR, ASW, LWK andYRI populations; thus in an embodiment, wherein the method involvesferroportin, eg administering an anti-ferroportin ligand (eg, antibody),the human is of AFR ancestry, eg, of, ASW, LWK or YRI ancestry.###rs7540883 is present in humans at an average cumulative frequency of2% according to the 1000 Genomes Phase I database, but is higher in AFR,ASW, LWK and YRI populations; thus in an embodiment, wherein the methodinvolves haemojuvelin, eg administering an anti-haemojuvelin ligand (eg,antibody or trap, such as human HJV-Fc dimer), the human is of AFRancestry, eg, of, ASW, LWK or YRI ancestry.

SNP rs111588693 encodes an amino acid in the region of the cleavage sitefor the proprotein of BM6, and thus it may be desirable to target a BMP6encoded by a nucleotide sequence comprising rs111588693 in the human,optionally wherein the human comprises a BMP6 nucleotide sequencecomprising said SNP.

In an embodiment, the human comprises a SNP selected from the SNPs incolumn 1 of Table 24; optionally also the ligand specifically binds aprotein (eg, a TOI of said humanhemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis) encodedby a nucleotide sequence comprising said SNP.

In an embodiment, the ligand (eg, trap, antibody or antibody fragment)specifically binds a TOI protein (eg, a TOI of said humanhemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis)comprising a corresponding variation as shown in Table 24 or is encodedby a nucleotide sequence comprising or encoding a SNP or amino acidvariation shown in Table 24.

For example, the TOI is a protein of the humanhemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis. In anexample, the TOI is human hemojuvelin. In an example, the TOI is humanBMP6 (and optionally comprising amino acid 28Q or encoded by a BMP6nucleotide sequence comprising SNP rs111588693; in an embodiment thehuman expresses BMP6 that comprises said variation). In an example, theTOI is human hepcidin. In an example, the TOI is human ferroportin. Inan example, the TOI is human transferrin (and optionally encoded by atransferrin nucleotide sequence comprising SNP rs3811647; in anembodiment the human expresses transferrin that comprises saidvariation). In an example, the TOI is human TMPRSS6 (and optionallycomprises one or more variations selected from the group consisting ofA736V, Y141C, I212T, R271Q, S304L and C510S; or is encoded by a TMPRSS6nucleotide sequence comprising SNP rs855791 or rs1421312 or encoding oneor more variations selected from said group; in an embodiment the humanexpresses TMPRSS6 that comprises said variation). For example, theTMPRSS6 comprises variation A736V and optionally the human expressesTMPRSS6 that comprises variation A736V. In an example, the TOI is humanerythroferrone. In an example, the TOI is human hemochromatosis protein,HFE (and optionally comprising amino acid 282Y or encoded by a HFEnucleotide sequence comprising SNP rs1800562; in an embodiment the humanexpresses HFE that comprises said variation). In an example, the TOI isthe product of the human CUBN nucleotide sequence or the human comprisesa CUBN nucleotide sequence, the nucleotide sequence comprising SNPrs10904850; in an embodiment the human expresses CUBN protein thatcomprises said variation. CUBN is also known as cubilin and intrinsicfactor-cobalamin receptor. Ferroportin is also known as solute carrierfamily 40 member 1 (SLC40A1) or iron-regulated transporter 1.Haemojuvelin is also known as hemojuvelin, HFE2A, HJV, JH and RGMC.

In an example, the human comprises SNP rs2698530 on chr. 2p14 and/orrs987710 on chr. 22q11.

It may be desirable to block (not necessarily clear as with an IgG1format) the TOI in the method of this alternative and a human IgG4 maybe preferable for doing this in an iron regulation setting. Thus, in anembodiment, the ligand comprises a human gamma-4 constant region. Forthe iron regulation setting of the invention, it may be advantageouswherein the gamma-4 is a gamma-4 constant region with 228Pro or 235Glu.In an example the constant region is an IgG4PE (ie, a gamma-4 constantregion with 228Pro and 235Glu). In an example, the ligand compriseshuman IGHG4*01 hinge S10>P constant region.

In an example, therefore, the ligand comprises any human gamma-4constant region disclosed herein (eg, in any of Examples 1 et seq).

References

-   (a) Nutr Hosp. 2012 November-December; 27(6):2142-5. doi:    10.3305/nh.2012.27.6.6154; “Intronic SNP rs3811647 of the human    transferrin gene modulates its expression in hepatoma cells”;    Blanco-Rojo R et al.    -   Transferrin (Tf) exerts a crucial function in the maintenance of        systemic iron homeostasis.    -   The expression of the Tf gene is controlled by transcriptional        mechanism. The authors conclude that A allele of SNP rs3811647        increases Tf expression in a manner that might underlie        inter-individual variation in serum transferrin levels observed        in different population groups.-   (b) J Med Genet. 2013 September; 50(9):593-8. doi:    10.1136/jmedgenet-2013-101673. Epub 2013 Jun. 21; “Associations of    common variants in HFE and TMPRSS6 with iron parameters are    independent of serum hepcidin in a general population: a replication    study”; Galesloot T E et al.    -   The authors found that HFE rs1800562 and TMPRSS6 rs855791 are        the main determinants of HFE and TMPRSS6 related variation in        serum iron, ferritin, transferrin saturation, and total iron        binding capacity.-   (c) BMC Nephrol. 2013 Feb. 22; 14:48. doi: 10.1186/1471-2369-14-48;    “The A736V TMPRSS6 polymorphism influences hepcidin and iron    metabolism in chronic hemodialysis patients: TMPRSS6 and hepcidin in    hemodialysis”; Pelusi S et al.    -   The A736V TMPRSS6 polymorphism is a major genetic determinant of        iron metabolism in healthy subjects. The authors reported the        TMPRSS6 736 V variant was associated with higher hepcidin levels        (p=0.017). In patients without acute inflammation and overt iron        deficiency (C reactive protein <1 mg/dl and ferritin >30 ng/ml;        n=86), hepcidin was associated with lower mean corpuscular        volume (p=0.002), suggesting that it contributed to        iron-restricted erythropoiesis. In line with previous results,        in patients without acute inflammation and severe iron        deficiency the “high hepcidin” 736 V TMPRSS6 variant was        associated with higher erythropoietin maintenance dose        (p=0.016), independently of subclinical inflammation (p=0.02).-   (d) PLoS One. 2012; 7(6):e38339. doi: 10.1371/journal.pone.0038339.    Epub 2012 Jun. 22;

“Associations between single nucleotide polymorphisms in iron-relatedgenes and iron status in multiethnic populations”; McLaren C E et al.

-   -   Three chromosomal regions showed association across multiple        populations, including SNPs in the TF and TMPRSS6 genes, and on        chromosome 18q21. A novel SNP rs1421312 in TMPRSS6 was        associated with serum iron in whites (p=3.7×10(−6)) and        replicated in African Americans (p=0.0012). Twenty SNPs in the        TF gene region were associated with total iron-binding capacity        in whites (p<4.4×10(−5)); six SNPs replicated in other        ethnicities (p<0.01). SNP rs10904850 in the CUBN gene on 10p13        was associated with serum iron in African Americans        (P=1.0×10(−5)).    -   (e) PLoS One. 2011 Mar. 31; 6(3):e17390. doi:        10.1371/journal.pone.0017390; “Genome-wide association study        identifies genetic loci associated with iron deficiency”;        McLaren C E et al. Five SNPs were identified in a Genome-Wide        Association Study that met genome-wide statistical significance        for association with at least one iron measure, rs2698530 on        chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the        transferrin (TF) gene region; rs1800562 on chr. 6p22, the C282Y        mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710        on chr. 22q11 (GWAS observed P<1.51×10(−7) for all). An        association was observed between total iron binding capacity and        SNP rs3811647 in the TF gene (GWAS observed P=7.0×10(−9). The        analysis revealed strong associations between rs2698530 on chr.        2p14 and iron status outcomes.    -   (f) Hum Mutat. 2010 May; 31(5):E1390-405. doi:        10.1002/humu.21243; “Novel TMPRSS6 mutations associated with        iron-refractory iron deficiency anemia (IRIDA)”; De Falco L et        al. Mutations leading to abrogation of matriptase-2 proteolytic        activity in humans are associated with an iron-refractory iron        deficiency anemia (IRIDA) due to elevated hepcidin levels. The        authors describe 12 IRIDA patients belonging to 7 unrelated        families and identify 10 (9 novel) TMPRSS6 mutations spread        along the gene sequence: 5 missense, 1 non sense and 4        frameshift. The frameshift and non sense mutations are predict        to result in truncated protein lacking the catalytic domain. The        causal role of missense mutations (Y141C, I212T, R271Q, 5304L        and C510S) is demonstrated by in silico analysis, their absence        in 100 control chromosomes and the high conservation of the        involved residues. The C510S mutation in the LDLRA domain in        silico model causes an intra-molecular structural imbalance that        impairs matriptase-2 activation. We also assessed the in vitro        effect on hepcidin promoter and the proteolytic activity of        I212T and R271Q variants demonstrating a reduced inhibitory        effect for the former mutation, but surprisingly a normal        function for R271Q which appears a silent mutation in vitro.        Based on mRNA expression studies I212T could also decrease the        total amount of protein produced, likely interfering with mRNA        stability. Collectively, our results extend the pattern of        TMPRSS6 mutations associated with IRIDA and propose a model of        causality for some of the novel missense mutation.

The invention provides the following concepts:—

-   1. A method of treating or reducing the risk of a disease or    condition in a human, wherein the disease or condition is mediated    by a TOI, the TOI being present in humans as a plurality of variants    differing by one or more amino acid polymorphisms, the method    comprising administering a ligand (eg, an antibody or antibody    fragment) to the human, the ligand comprising first and second    protein domains, wherein the first domain specifically binds a TOI    variant comprising a first amino acid polymorphism, wherein the    second domain comprises a second polymorphism, and wherein the human    expresses (i) TOI comprising said first amino acid polymorphism;    and (ii) protein domains comprising said second polymorphism,    optionally wherein the ligand comprises a human gamma-4 heavy chain    constant region that comprises a Leu at position 189 shown in SEQ ID    NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein    said human comprises an IGHG4*01 human heavy chain constant region    gene segment, or the human expresses antibodies comprising human    gamma-4 heavy chain constant regions comprising a Leu at position    189 shown in SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID    NO: 73; and wherein the second domain is comprised by said gamma-4    heavy chain constant region of the ligand.    -   Optionally, the TOI is selected from the human        hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.        In an example, the TOI is human hemojuvelin. In an example, the        TOI is human BMP6. In an example, the TOI is human TMPRSS6. In        an example, the TOI is human hepcidin. In an example, the TOI is        human ferroportin. In an example, the TOI is human HFE. In an        example, the TOI is human transferrin. A TOI of “the human        hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis”        is a human protein (eg, expressed by liver) that is part of the        iron regulation pathway comprising hemojuvelin, BMP6, TMPRSS6,        hepcidin, ferroportin and transferrin.    -   For example, the method can be for any of the purposes or        indications set out section A. In an example, the method is for        regulating iron in the human. In an example, the iron is blood        iron or circulating iron or free iron.    -   In an example, the method is a method of reducing anaemia in the        human.-   2. The method of concept 1, wherein the first and/or second    polymorphism is respectively encoded by a SNP having a cumulative    human allele frequency of less than 50%.    -   Optionally the frequency is less than 45, 40, 35, 30, 25, 20,        15, 10, 5, 4, 3, 2, or 1%.-   3. The method of any preceding concept, wherein the first domain is    an antibody variable domain or a receptor domain.-   4. The method of any preceding concept, wherein the second domain is    an antibody domain, optionally an antibody variable or constant    domain.-   5. The method of any preceding concept, wherein the second domain is    a domain of an antibody Fc region.-   6. The method of any one of concepts 1 to 4, wherein the first and    second domains are antibody variable domains, optionally wherein the    variable domains specifically bind first and second human TOI    respectively, wherein the TOIs are different.    -   Optionally, the second TOI is selected from the human        hemojuvelin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin axis.        In an example, the second TOI is human hemojuvelin. In an        example, the second TOI is human BMP6. In an example, the second        TOI is human TMPRSS6. In an example, the second TOI is human        hepcidin. In an example, the second TOI is human ferroportin. In        an example, the second TOI is human HFE. In an example, the        second TOI is human transferrin.-   7. The method of any one of concepts 1 to 4, wherein the first and    second domains are receptor domains, optionally wherein the receptor    domains specifically bind first and second human TOI respectively,    wherein the TOIs are different.-   8. The method of concept 6 or 7, wherein the first domain comprises    a third amino acid polymorphism, wherein the human expresses (iii)    domains comprising said third amino acid polymorphism.    -   Optionally the human expresses the second TOI variant.    -   Optionally the third polymorphism is encoded by a SNP having a        cumulative human frequency of less than 50%. Optionally the        frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3,        2, or 1%.    -   In an example, the first domain is an antibody variable domain        (eg, any V domain of the invention described herein, such human        VH3-23*04 as herein described) and the domains of (iii) are        antibody variable domains. For example, the first domains and        domains of (iii) are VH domains. For example, the first domains        and domains of (iii) are Vκ domains. For example, the first        domains and domains of (iii) are Vλ, domains.    -   In an example, the first domain is a receptor binding site and        the domains of (iii) are said receptor binding site domains.-   9. A method according to concepts 1, 2 and 8 combined.-   10. The method of any preceding concept, wherein the human expresses    the first and/or second domain.-   11. The method of any preceding concept, wherein the first, second    and/or third polymorphism is respectively found in at least 5    different human populations as defined in Table 4.    -   For example, each of the first and second polymorphisms is        respectively found in at least 5 different human populations as        defined in Table 4. For example, each of the first, second and        polymorphisms is respectively found in at least 5 different        human populations as defined in Table 4.    -   Optionally the first, second and/or third (see below)        polymorphism respectively appears in at least 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19 or 20 different human populations as        defined in Table 4.-   12. The method of concept 11, wherein the first, second and/or third    polymorphism respectively appears in at least 15 humans in the 1000    Genomes Phase I database.    -   In an embodiment, the first, second and/or third polymorphism is        found in at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,        75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150        human individuals distributed across such many different human        populations.-   13. The method of any preceding concept, wherein the first, second    and/or third polymorphism is respectively found in at least 5    different human populations as defined in Table 4 and is encoded by    a SNP having a cumulative human allele frequency of less than 50%.    -   Optionally the frequency is less than 45, 40, 35, 30, 25, 20,        15, 10, 5, 4, 3, 2, or 1%.-   14. The method of any preceding concept, wherein the first, second    and/or third polymorphism is respectively encoded by a SNP having    total human genotype frequency of less than 50%.    -   Optionally the frequency is less than 45, 40, 35, 30, 25, 20,        15, 10, 5, 4, 3, 2, or 1%.-   15. The method of any preceding concept, wherein the first and    second domains are human, optionally wherein the ligand is fully    human.-   16. The method of any preceding concept, wherein the ligand is an    anti-TOI trap, antibody or antibody fragment.-   17. The method of any preceding concept, wherein the ligand    specifically binds two or more human TOIs, eg, said first and second    TOIs.-   18. The method of any preceding concept, wherein the ligand is    administered to the human by injection or the ligand is provided by    an injectable preparation.-   19. The method of any preceding concept, wherein said human has been    or is genotyped as positive for said TOI variant nucleotide sequence    before administering the ligand, or the method comprises genotyping    the human as positive for said variant nucleotide sequence before    administering the ligand.-   20. The method of any preceding concept, wherein the human has been    or is phenotyped as positive for said TOI variant before    administering the ligand, or the method comprises phenotyping the    human as positive for said variant before administering the ligand.-   21. The method of any preceding concept, comprising, before said    administering, selecting a human comprising said first TOI    polymorphism, wherein the human is the human of concept 1.-   22. The method of any preceding concept, wherein the human has been    determined to comprise the first and/or second TOI variant or a    nucleotide sequence encoding the TOI variant(s).-   23. The method of any preceding concept, comprising the step of    determining that the human comprises a first TOI nucleotide sequence    encoding the first polymorphism, optionally, wherein the determining    step is performed before administration of the ligand to the human.-   24. The method of concept 23 wherein the step of determining    comprises assaying a biological sample from the human for said    nucleotide sequence.-   25. The method of concept 24, wherein the assaying comprises    contacting the biological sample with    -   h. a. at least one oligonucleotide probe comprising a sequence        of at least 10 contiguous nucleotides that can specifically        hybridize to and identify in the biological sample a nucleotide        sequence comprising a SNP encoding the first polymorphism or        that specifically hybridizes to an antisense of said sequence,        wherein said nucleic acid hybridizes to said SNP or hybridizes        to an antisense sequence thereby forming a complex when at least        one nucleotide sequence comprising said SNP is present; and/or    -   i. b. at least one oligonucleotide probe comprising a sequence        of at least 10 contiguous nucleotides of a nucleotide sequence        comprising said SNP or comprising an antisense sequence of said        contiguous nucleotides, wherein said sequence of contiguous        nucleotides comprises said SNP thereby forming a complex when        the nucleotide sequence comprising said SNP is present; and    -   j. detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the first TOI nucleotide sequence.-   26. The method of concept 24, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   27. The method of any preceding concept, wherein said human is or    has been further determined to be substantially resistant to an    anaemia treatment.-   28. The method of any preceding concept, wherein said human is    receiving or has received an anaemia treatment or has reduced    responsiveness to an anaemia treatment.-   29. The method of concept 25, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   30. The method of any preceding concept, wherein said human has been    diagnosed with said disease or condition.-   31. An anti-TOI ligand (eg, an antibody, antibody fragment or trap)    for use in the method of any preceding concept.    -   In an embodiment, the trap is a receptor Fc fusion (ie, a        receptor domain fused to an antibody Fc region, eg, as a dimer),        wherein the receptor specifically binds to the TOI.-   32. The ligand of concept 31, wherein the ligand is fully human.-   33. The ligand of concept 31 or 32, wherein the ligand is for    administration by injection or the ligand is provided by an    injectable preparation.

In an example, the first, second and/or third polymorphismrespectively:—

-   -   k. has a cumulative human allele frequency of 15% or less;    -   l. has a total human genotype frequency of 20% or less;    -   m. is found in at least 5 different human populations (using the        standard categorisation of the 1000 Genomes Project, eg, as per        the Phase I database); and    -   n. is found in many individuals distributed across such many        different populations.

In an example, the criteria are applied with reference to one or morehuman genomic sequence databases as described herein. For example, thecriteria are those as applied to the 1000 Genomes database.

For example in any aspect example, embodiment or configuration of theinvention, the 1000 Genomes database release 13 or Phase I. For example,the 1000 Genomes database in its most recent version as at 1 Oct. 2013.

In an example, the human is homozygous for the first polymorphism.Additionally or alternatively, the human is homozygous for the firstpolymorphism. Additionally or alternatively, the human is homozygous forthe third polymorphism. For example, the human is homozygous for thefirst and second polymorphisms.

In an example, the method is according to any one of the disclosureherein involving selecting, genotyping or phenotyping the human aspositive for the TOI variant (ie, in the present case human PD-1comprising the polymorphism or a nucleotide sequence encoding this). Inan example, the method is according to any one of concepts aboveinvolving the determining step or wherein the human has been determinedto comprise the variant TOI variant (eg, in the present case human PD-1comprising the polymorphism or a nucleotide sequence encoding this).

In an example, the first polymorphism is a human BMP6 amino acidpolymorphism described herein or is encoded by a human BMP6 SNPdescribed herein.

In an example, wherein the TOI is human BMP6 the first polymorphism isencoded by SNP rs111588693.

The invention further provides the following specific aspects:—

-   -   1. A method of treating or reducing the risk of anaemia in a        human by targeting in the human a human TOI selected from the        group consisting of BMP6, ferroportin, haemojuvelin, hepcidin        and TMPRSS6, the method comprising administering an antibody or        antibody fragment to the human, wherein the antibody or antibody        fragment specifically binds a variant of said TOI comprising a        first amino acid polymorphism, wherein the antibody or antibody        fragment comprises a human gamma-4 heavy chain constant region        that comprises a Leu at position 189 shown in SEQ ID NO: 73 or        an Arg at position 289 shown in SEQ ID NO: 73 and wherein said        human comprises (i) an IGHG4*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-4 heavy chain constant regions comprising        a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at        position 289 shown in SEQ ID NO: 73; and (ii) a PD-1 nucleotide        sequence encoding a PD-1 comprising said first amino acid        polymorphism or the human expresses a PD-1 comprising said first        amino acid polymorphism.    -   Preferably, the trap, antibody or fragment of any configuration,        concept, aspect, example or embodiment comprises an IgG4PE        constant region.    -   In an example of any configuration, concept, aspect, example or        embodiment, an anti-TOI (eg, anti-BMP6) antibody is used.    -   A first aspect provides:—    -   A method of treating or reducing the risk of anaemia in a human,        the method comprising administering an anti-BMP6 trap, antibody        or antibody fragment to the human, wherein the trap, antibody or        antibody fragment specifically binds a human BMP6 encoded by a        BMP6 nucleotide sequence comprising a SNP rs111588693, wherein        the trap, antibody or antibody fragment comprises a human        gamma-4 heavy chain constant region that comprises a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73 and wherein said human comprises (i) an        IGHG4*01 human heavy chain constant region gene segment, or the        human expresses antibodies comprising human gamma-4 heavy chain        constant regions comprising a Leu at position 189 shown in SEQ        ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73;        and (ii) a BMP6 nucleotide sequence comprising said SNP;        optionally wherein the human has been determined to comprise        said SNP or the method comprises determining that the human        comprises said SNP before administering the antibody or        fragment.    -   A second aspect provides:—    -   A method of treating or reducing the risk of anaemia in a human,        the method comprising administering an anti-haemojuvelin trap,        antibody or antibody fragment to the human, wherein the trap,        antibody or antibody fragment specifically binds a human        haemojuvelin encoded by a haemojuvelin nucleotide sequence        comprising SNP rs7540883, wherein the trap, antibody or antibody        fragment comprises a human gamma-4 heavy chain constant region        that comprises a Leu at position 189 shown in SEQ ID NO: 73 or        an Arg at position 289 shown in SEQ ID NO: 73 and wherein said        human comprises (i) an IGHG4*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-4 heavy chain constant regions comprising        a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at        position 289 shown in SEQ ID NO: 73; and (ii) a haemojuvelin        nucleotide sequence comprising said SNP; optionally wherein the        human has been determined to comprise said SNP or the method        comprises determining that the human comprises said SNP before        administering the antibody or fragment.    -   A third aspect provides:—    -   A method of treating or reducing the risk of anaemia in a human,        the method comprising administering an anti-ferroportin trap,        antibody or antibody fragment to the human, wherein the trap,        antibody or antibody fragment specifically binds a human        ferroportin encoded by a ferroportin nucleotide sequence        comprising SNP rs11568350, wherein the trap, antibody or        antibody fragment comprises a human gamma-4 heavy chain constant        region that comprises a Leu at position 189 shown in SEQ ID NO:        73 or an Arg at position 289 shown in SEQ ID NO: 73 and wherein        said human comprises (i) an IGHG4*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-4 heavy chain constant regions comprising        a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at        position 289 shown in SEQ ID NO: 73; and (ii) a ferroportin        nucleotide sequence comprising said SNP; optionally wherein the        human has been determined to comprise said SNP or the method        comprises determining that the human comprises said SNP before        administering the antibody or fragment.    -   A fourth aspect provides:—    -   A method of treating or reducing the risk of anaemia in a human,        the method comprising administering an anti-TMPRSS6 trap,        antibody or antibody fragment to the human, wherein the trap,        antibody or antibody fragment specifically binds a human TMPRSS6        encoded by a TMPRSS6 nucleotide sequence comprising a SNP        selected from the group consisting of rs855791, rs1421312,        rs2543519, rs2235324 wherein the trap, antibody or antibody        fragment comprises a human gamma-4 heavy chain constant region        that comprises a Leu at position 189 shown in SEQ ID NO: 73 or        an Arg at position 289 shown in SEQ ID NO: 73 and wherein said        human comprises (i) an IGHG4*01 human heavy chain constant        region gene segment, or the human expresses antibodies        comprising human gamma-4 heavy chain constant regions comprising        a Leu at position 189 shown in SEQ ID NO: 73 or an Arg at        position 289 shown in SEQ ID NO: 73; and (ii) a TMPRSS6        nucleotide sequence comprising said selected SNP; optionally        wherein the human has been determined to comprise said SNP or        the method comprises determining that the human comprises said        SNP before administering the antibody or fragment.    -   A fifth aspect provides:—    -   A method of treating or reducing the risk of anaemia in a human,        the method comprising administering an anti-hepcidin trap,        antibody or antibody fragment to the human, wherein the trap,        antibody or antibody fragment specifically binds a human        hepcidin encoded by a hepcidin nucleotide sequence comprising        SNP rs146776859, wherein the trap, antibody or antibody fragment        comprises a human gamma-4 heavy chain constant region that        comprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg        at position 289 shown in SEQ ID NO: 73 and wherein said human        comprises (i) an IGHG4*01 human heavy chain constant region gene        segment, or the human expresses antibodies comprising human        gamma-4 heavy chain constant regions comprising a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73; and (ii) a hepcidin nucleotide sequence        comprising said SNP; optionally wherein the human has been        determined to comprise said SNP or the method comprises        determining that the human comprises said SNP before        administering the antibody or fragment.    -   2. The method of aspect 1, wherein the human is homozygous for        said selected SNP.    -   3. The method of any preceding aspect, wherein the ligand        comprises an IGHG4*01 human heavy chain constant region, eg,        wherein the constant region is a gamma-4 constant region with        228Pro and/or 235Glu.    -   4. The method of any preceding aspect, wherein the anaemia is        selected from the group consisting of Anaemia of Chronic        Inflammation (ACI); Anaemia of Chronic Disease (ACD); or anaemia        associated with cancer, a kidney condition or GvHD.    -   5. The method of any preceding aspect, comprising, before said        administering, selecting a human comprising said BMP6,        ferroportin, haemojuvelin, TMPRSS6 or hepcidin nucleotide        sequence of (ii), wherein the human is the human of aspect 1.    -   6. The method of any preceding aspect, wherein the human has        been determined to comprise said BMP6, ferroportin,        haemojuvelin, TMPRSS6 or hepcidin nucleotide sequence of (ii).    -   7. The method of any preceding aspect, comprising the step of        determining that the human comprises said BMP6, ferroportin,        haemojuvelin, TMPRSS6 or hepcidin nucleotide sequence of (ii),        optionally, wherein the determining step is performed before        administration of the antibody to the human.    -   8. The method of aspect 7, wherein the step of determining        comprises assaying a biological sample from the human for        respectively a BMP6, ferroportin, haemojuvelin, TMPRSS6 or        hepcidin nucleotide sequence comprising said selected variation.    -   9. The method of aspect 8, wherein the assaying comprises        contacting the biological sample with        -   a. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides that can specifically            hybridize to and identify in the biological sample a            nucleotide sequence comprising said selected variation or            that specifically hybridizes to an antisense of said            sequence, wherein said nucleic acid hybridizes to said            selected SNP or hybridizes to an antisense sequence thereby            forming a complex when at least one nucleotide sequence            comprising said selected SNP is present; and/or        -   b. at least one oligonucleotide probe comprising a sequence            of at least 10 contiguous nucleotides of a nucleotide            sequence comprising said selected SNP or comprising an            antisense sequence of said contiguous nucleotides, wherein            said sequence of contiguous nucleotides comprises said            selected SNP thereby forming a complex when the nucleotide            sequence comprising said selected SNP is present; and            -   c. detecting the presence or absence of the complex,                wherein detecting the presence of the complex determines                that the human comprises respectively a BMP6,                ferroportin, haemojuvelin, TMPRSS6 or hepcidin                nucleotide sequence comprising said selected SNP.    -   10. The method of aspect 9, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   11. The method of any preceding aspect, wherein said human is or        has been further determined to be substantially resistant an        anaemia treatment (eg, EPO).        -   For example the human is or has been further determined to            be resistant to an anaemia treatment (eg, EPO).    -   12. The method of any preceding aspect, wherein said human is        receiving or has an anaemia treatment (eg, EPO) or has reduced        responsiveness to an anaemia treatment (eg, EPO).    -   13. The method of aspect 8, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.    -   14. The method of any preceding aspect, wherein said trap,        antibody or fragment is administered by intravenous or        subcutaneous injection and/or is comprised in an injectable        preparation.    -   15. The method of any preceding aspect, wherein the trap,        antibody or fragment is human, eg, a human antibody or antibody        fragment.    -   16. An anti-human TOI trap, antibody or antibody fragment for        use in the method of any preceding aspect, wherein the TOI is        selected from BMP6, ferroportin, haemojuvelin, TMPRSS6 and        hepcidin.        -   In an embodiment, the trap is a haemojuvelin- or receptor-Fc            fusion (ie, a receptor domain fused to an antibody Fc            region, eg, as a dimer), wherein the receptor specifically            binds to or competes with a TOI is selected from BMP6,            ferroportin, haemojuvelin, TMPRSS6 and hepcidin.    -   17. The ligand of aspect 17, wherein the trap, antibody or        antibody fragment is fully human.    -   18. The ligand of aspect 16 or 17, wherein the trap, antibody or        antibody fragment is for administration by injection or the        trap, antibody or antibody fragment is provided by an injectable        preparation.

Optionally in any of the configurations, concepts, aspects, embodimentsor examples, one or more of the following optional features applies.

Optionally, the ligand (eg, trap, antibody or antibody fragment) isaccording to one of the following options:—

(i) wherein the ligand comprises a VH domain derived from therecombination of a human VH segment (eg, human VH3-23*04), a human Dgene segment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40.(ii) wherein the ligand comprises a VH domain derived from therecombination of human VH segment selected from the group consisting ofIGHV3-33*01 and IGHV3-7*01, a human D gene segment and a human JHsegment, and wherein said human comprises said selected VH gene segmentor the human expresses VH domains derived from the recombination of saidselected human VH segment, a human D gene segment and a human JHsegment.(iii) wherein the ligand comprises a Vκ domain derived from therecombination of human Vκ segment selected from the group consisting ofIGKV3-11*01 and IGKV1-12*01 and a human Jκ segment, and wherein saidhuman comprises said selected Vκ gene segment or the human expresses Vκdomains derived from the recombination of said selected human Vκ segmentand a human Jκ segment.(iv) wherein the ligand comprises a Vκ domain derived from therecombination of a human Vκ segment and a human Jκ segment, the human Vκsegment encoding (i) a CDR3 comprising a Pro at position 7 shown in SEQID NO: 36 and wherein said human comprises a Vκ gene segment encoding aCDR3 comprising a Pro at position 7 shown in SEQ ID NO: 36, or the humanexpresses Vκ domains that comprise a CDR3 comprising a Pro at position 7shown in SEQ ID NO: 36; or (ii) a FW3 comprising a Ser at position 15shown in SEQ ID NO: 38 and wherein said human comprises a Vκ genesegment encoding a FW3 comprising a Ser at position 15 shown in SEQ IDNO: 38 or the human expresses Vκ domains that comprise a FW3 comprisinga Ser at position 15 shown in SEQ ID NO: 38.

Optionally, instead of a gamma-4 constant region, the ligand (eg, atrap, antibody or fragment) comprises a heavy chain constant regionaccording to any of the following options:—

(v) wherein the ligand comprises a human gamma-1 heavy chain constantregion that comprises an Asp at position 204 shown in SEQ ID NO: 4 or aLeu at position 206 shown in SEQ ID NO: 4 and wherein said humancomprises (i) an IGHG1*01 human heavy chain constant region genesegment, or the human expresses antibodies comprising human gamma-1heavy chain constant regions comprising an Asp at position 204 shown inSEQ ID NO: 4 or a Leu at position 206 shown in SEQ ID NO: 4.(vi) wherein the ligand comprises a human gamma-2 heavy chain constantregion that comprises an amino acid selected from the group consistingof a Pro at position 72 shown in SEQ ID NO: 6, an Asn at position 75shown in SEQ ID NO: 6, a Phe at position 76 shown in SEQ ID NO: 6, a Valat position 161 shown in SEQ ID NO: 6 and an Ala at position 257 shownin SEQ ID NO: 6 and wherein said human comprises (i) an IGHG2*01 humanheavy chain constant region gene segment, or the human expressesantibodies comprising human gamma-2 heavy chain constant regionscomprising said selected Pro at position 72 shown in SEQ ID NO: 6, Asnat position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ IDNO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257shown in SEQ ID NO: 6. In an example, the constant region is Fcenhanced, eg, ADCC or CDC enhanced. The skilled person will knowtechniques (eg, known C region mutations) to enhance ADCC or CDC.(vii) wherein the ligand comprises a human gamma-3 heavy chain constantregion encoded by a first human IGHG3 (eg, IGHG3*01) constant regiongene segment and wherein said human comprises (i) said first constantregion gene segment (eg, an IGHG3*01), or the human expresses antibodiescomprising human gamma-3 heavy chain constant regions encoded by saidfirst human IGHG3 (eg, IGHG3*01) constant region gene segment.

Optionally, the ligand (eg, an antibody or antibody fragment) isaccording to one of the following options:—

(viii) wherein the ligand comprises a human kappa chain constant regionthat comprises a Val at position 84 shown in SEQ ID NO: 16 or a Cys atposition 87 shown in SEQ ID NO: 16 and wherein said human comprises (i)an IGKC1*01 human kappa chain constant region gene segment, or the humanexpresses antibodies comprising human kappa chain constant regionscomprising a Val corresponding to position 84 shown in SEQ ID NO: 16 ora Cys at position 87 shown in SEQ ID NO: 16.(ix) wherein the ligand comprises a human IGLC1*01 lambda chain constantregion and wherein said human comprises (i) a human IGLC1*01 lambdachain constant region gene segment, or the human expresses antibodiescomprising human IGLC1*01 lambda chain constant regions.(x) wherein the ligand comprises a human epsilon heavy chain constantregion encoded by a first human epsilon heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human epsilonheavy chain constant regions encoded by said first constant region genesegment.(xii) wherein the ligand comprises a human mu heavy chain constantregion encoded by a first human mu heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human muheavy chain constant regions encoded by said first constant region genesegment.(xiii) wherein the ligand comprises a human alpha heavy chain constantregion encoded by a first human alpha heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human alphaheavy chain constant regions encoded by said first constant region genesegment.(xiv) wherein the ligand comprises a human delta heavy chain constantregion encoded by a first human delta heavy chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human deltaheavy chain constant regions encoded by said first constant region genesegment.(xv) wherein the ligand comprises a human kappa light chain constantregion encoded by a first human kappa light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human kappalight chain constant regions encoded by said first constant region genesegment.(xvi) wherein the ligand comprises a human lambda light chain constantregion encoded by a first human lambda light chain constant region genesegment and wherein said human comprises (i) said first constant regiongene segment, or the human expresses antibodies comprising human lambdalight chain constant regions encoded by said first constant region genesegment.

Example 9 Nav1.8 (SCN10A; PN3; SNS)

Nav1.8 is a sodium ion channel that in humans is encoded by the SCN10Agene (OMIM: 604427). Nav1.8 is a tetrodotoxin (TTX)-resistantvoltage-gated sodium ion channel. It is expressed in the dorsal rootganglion (DRG), in unmyelinated, small-diameter sensory neurons calledC-fibres and is involved in the pain pathway (nociception). C-fibres canbe activated by noxious thermal or mechanical stimuli and thus can carrypain messages. It is known that Nav1.7, Nav1.8 and Nav1.9 are located inthe DRG and play an important role in maintaining chronic inflammatorypain. Nav1.8 α-subunit consists of four homologous domains each with sixtransmembrane spanning regions of which one is a voltage sensor. Nav1.8contributes the most to sustaining the depolarising stage of the actionpotentials in nociceptive sensory neurons by activating quickly andremaining activated after detecting a noxious stimuli. Therefore, Nav1.8is known to perform a key role in hyperalgesia (increased sensitivity topain) and allodynia (pain from stimuli that do not usually cause it)which are part of chronic pain. Nav1.8 knockout mice studies have shownthat the channel is associated with inflammatory and neuropathic pain.Moreover, Nav1.8 plays a crucial role in cold pain. Mutations in SCN10Aare associated to Brugada Syndrome.

Human Nav1.8 polymorphism has been observed. Reference is made to thefollowing, the disclosures of which are incorporated herein by referencein their entirety:—

-   1. Waxman S G et al, Lancet Neurol. 2014 November; 13(11):1152-60.    doi: 10.1016/S1474-4422(14)70150-4; “Sodium channel genes in    pain-related disorders: phenotype-genotype associations and    recommendations for clinical use”.-   2. Hoeijmakers J G et al, “Neurosci Lett. 2014 Dec. 31. pii:    50304-3940(14)01009-X. doi: 10.1016/j.neulet.2014.12.056; “Painful    peripheral neuropathy and sodium channel mutations”.-   3. Huang J et al, J Neurosci. 2013 Aug. 28; 33(35):14087-97. doi:    10.1523/JNEUROSCI.2710-13.2013; “Small-fiber neuropathy Nav1.8    mutation shifts activation to hyperpolarized potentials and    increases excitability of dorsal root ganglion neurons”.-   4. Han C et al, J Neurol Neurosurg Psychiatry. 2014 May;    85(5):499-505. doi: 10.1136/jnnp-2013-306095. Epub 2013 Sep. 4; “The    G1662S NaV1.8 mutation in small fibre neuropathy: impaired    inactivation underlying DRG neuron hyperexcitability”.-   5. Faber C et al, Proc Natl Acad Sci USA. 2012 Nov. 20;    109(47):19444-9. doi: 10.1073/pnas.1216080109. Epub 2012 Oct. 31;    “Gain-of-function Nav1.8 mutations in painful neuropathy”.-   6. Chambers J C et al, Nat Genet. 2010 February; 42(2):149-52. doi:    10.1038/ng.516. Epub 2010 Jan. 10; “Genetic variation in SCN10A    influences cardiac conduction”.-   7. Arisawa T et al, J Gastroenterol. 2013 January; 48(1):73-80. doi:    10.1007/s00535-012-0602-3. Epub 2012 May 23; “Genetic polymorphisms    of SCN10A are associated with functional dyspepsia in Japanese    subjects”.-   8. van den Boogaard M et al, J Clin Invest. 2014 April;    124(4):1844-52. doi: 10.1172/JCI73140. Epub 2014 Mar. 18; “A common    genetic variant within SCN10A modulates cardiac SCN5A expression”.-   9. U.S. Pat. No. 7,582,745: “Compositions and methods for inhibiting    expression of Nav1.8 gene”.-   10. Jabbari J et al, Circ Cardiovasc Genet. 2015 February;    8(1):64-73. doi: 10.1161/HCG.0000000000000022; “Common and Rare    Variants in SCN10A Modulate the Risk of Atrial Fibrillation”.

Mutations in the SCN10A gene account for approximately 5 percent ofcases of small fibre neuropathy, a condition characterized by severepain attacks and a reduced ability to differentiate between hot andcold. The mutations cause amino acid change in the NaV1.8 sodium channelMany of the mutations result in NaV1.8 sodium channels that open moreeasily than usual, increasing the flow of sodium ions that produce nerveimpulses within nociceptors. This increase in sodium ions enhancestransmission of pain signals, causing individuals to be more sensitiveto stimulation that might otherwise not cause pain. In this condition,the small fibres that extend from the nociceptors and transmit painsignals (axons) degenerate over time. The cause of this degeneration isunknown, but it likely accounts for signs and symptoms such as the lossof temperature differentiation.

Painful peripheral neuropathies or small-fibre neuropathies aredisorders of unmyelinated nociceptive C-fibres causing neuropathic painand in some cases there is no known cause. A gain-of-function mutationwas found in the Nav1.8 encoding SCN10A gene in patients with painfulperipheral neuropathy (Faber et al). Faber et al used voltage clamp andcurrent clamp methods along with predictive algorithms on 104 idiopathicpatients who did not have the mutation in SCN9A (encoding Nav1.7). Theyfound two gain-of-function mutations in SCN10A in three patients inwhich both mutations cause increased excitability in DRG sensory neuronsand hence they contribute to pain.

Certain common variants (polymorphisms) in the SCN10A gene have beenfound to increase the risk of developing an irregular heartbeat(arrhythmia). These polymorphisms lead to the production of an alteredNaV1.8 sodium channel that can disrupt the electrical signals thatcontrol the heartbeat. Specifically, changes in the SCN10A gene areassociated with a type of arrhythmia known as heart block. Heart blockoccurs when the heart's electrical signals are slowed down orinterrupted. It is unknown how changes to the NaV1.8 sodium channel leadto heart block.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.8-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.8protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.8 ligands; and Nav1.8-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of Nav1.8, in particular human Nav1.8 or its ligands and inscreening assays to identify other antagonists of Nav1.8 activity. Someof the ligands of the invention are useful for inhibitingNav1.8-mediated activities.

Anti-Nav1.8 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human Nav1.8, which is a commonly-occurring form (see, eg, SEQ ID NO:135). While such ligands may be useful for human patients harbouringthis form of human Nav1.8, the inventor considered it useful toinvestigate the possibility of targeting rarer—but stillnaturally-occurring—forms of Nav1.8 amongst human populations. In thisway, the inventor arrived at insight into the natural occurrences anddistributions of rarer human Nav1.8 forms that can serve as usefultargets (at the protein or nucleic acid level) for human treatment,prophylaxis and diagnosis pertinent to diseases and conditions mediatedor associated with Nav1.8 activity. This particularly provides fortailored therapies, prophylaxis and diagnosis in humans that are devoidof the common Nav1.8 gene or protein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer Nav1.8 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous (aka“missense”) changes at the nucleotide level that translate into one ormore corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor saw that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-Nav1.8 ligand for administration to human patientsfor therapy and/or prophylaxis of Nav1.8-mediated or associated diseasesor conditions. In this way, the patient receives drugs and ligands thatare tailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example a set of humanNav1.8 variants can be determined on the basis of the followingcriteria, these being criteria that the inventor realised would providefor useful medical drugs and diagnostics to tailored need in the humanpopulation. The inventor selected some variants having at least 3 of the4 following criteria:—

Naturally-occurring human Nav1.8 variation having a cumulative humanallele frequency of 35% or less;

Naturally-occurring human Nav1.8 variation having a total human genotypefrequency of about 50% or less;

Naturally-occurring human Nav1.8 variation found in many different humanethnic populations (using the standard categorisation of the 1000Genomes Project; see Table 2 below); and

Naturally-occurring human Nav1.8 variation found in many individualsdistributed across such many different ethnic populations.

The selection can include, as an additional or alternativeconsideration, selection for nucleotide variation that produced aminoacid variation in corresponding Nav1.8 forms (ie, non-synonymousvariations), as opposed to silent variations that do not alter aminoacid residues in the target protein.

Variants can also be sourced from Ensembl, 1000 Genomes database or theliterature.

The inventor identified variants listed in Table 25 below.

TABLE 25 Nav 1.8 - Human Polymorphism & SNPs CHROMOSOMAL LOCATION AMINOAMINO (FORWARD NUCLEOTIDE ACID ACID CUMULATIVE EXAMPLE SNP/VARIANTSTRAND) CHANGE CHANGE POSITION FREQUENCY¹ CONDITIONS rs1384047833:38752313 1661T>C L554P 554 Pain Inflammatory pain Neuropathic pain,eg, SFN 3910G>A A1304T 1304 Pain Inflammatory pain Neuropathic pain, eg,SFN 4984G>A 11706V 1706 Pain Inflammatory pain Neuropathic pain, eg, SFN2884A>G Dyspepsia EPS DPS Gastric pain 3218C>T Dyspepsia EPS DPS Gastricpain *rs6801957 3:38725824 C>T 0.305 Brugada Syndrome Cardiac painCardiac arrhythmia Heart block Ventricular fibrillation Atrialfibrillation **rs6795970 3:38725184 G>A A1073V 1073 0.264 Cardiac painCardiac arrhythmia Heart block Ventricular fibrillation Atrialfibrillation G1662S 1662 Pain Inflammatory pain Neuropathic pain, eg,SFN ***rs10428132 3:38736063 G>T 0.296 Brugada Syndrome Cardiac painCardiac arrhythmia Heart block Ventricular fibrillation Atrialfibrillation ****rs12632942 3:38723507 A>G L1092P 1092 0.237 PainInflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiacpain Cardiac arrhythmia Heart block Ventricular fibrillation Atrialfibrillation *****rs57326399 3:38726809 T>C 1962V 962 0.231 PainInflammatory pain Neuropathic pain, eg, SFN Brugada Syndrome Cardiacpain Cardiac arrhythmia Heart block Ventricular fibrillation Atrialfibrillation +rs7630989 3:38752449 A>G 5509P 509 0.137 Pain Inflammatorypain Neuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiacarrhythmia Heart block Ventricular fibrillation Atrial fibrillation++rs74717885 3:38763578 T>C 1206M 206 0.048 Pain Inflammatory painNeuropathic pain, eg, SFN Brugada Syndrome Cardiac pain Cardiacarrhythmia Heart block Ventricular fibrillation Atrial fibrillation+++rs144270136 3:38793743 G>A R90W 90 0.009 Neuropathic pain, eg, SFNBrugada Syndrome Cardiac pain Cardiac arrhythmia Heart block Ventricularfibrillation Atrial fibrillation SFN = small fibre neuropathy or painassociated with small fibre neuropathy EPS = Epigastric pain syndromePDS = Postprandial distress syndrome ¹According to Ensembl, dbSNP*rs6801957 is present in humans at an average cumulative frequency of31% according to the 1000 Genomes Phase I database, but is higher inAMR, EUR, CLM, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus inan embodiment, in the present invention involving Nav 1.8 the human isof AMR ancestry, eg, of CLM, MXL or PUR ancestry; or of EUR ancestry,eg, of CEU, FIN, GBR, IBS or TSI ancestry. **rs6795970 is present inhumans at an average cumulative frequency of 26% according to the 1000Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL, PUR, CEU,FIN, GBR, IBS and TSI populations; thus in an embodiment, in the presentinvention involving Nav 1.8 the human is of AMR ancestry, eg, of CLM,MXL or PUR ancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS orTSI ancestry. ***rs10428132 is present in humans at an averagecumulative frequency of 30% according to the 1000 Genomes Phase Idatabase, but is higher in AMR, EUR, CLM, MXL, PUR, CEU, FIN, GBR, IBSand TSI populations; thus in an embodiment, in the present inventioninvolving Nav 1.8 the human is of AMR ancestry, eg, of CLM, MXL or PURancestry; or of EUR ancestry, eg, of CEU, FIN, GBR, IBS or TSI ancestry.****rs12632942 is present in humans at an average cumulative frequencyof 24% according to the 1000 Genomes Phase I database, but is higher inASN, EUR, CHB, CHS, JPT, IBS and TSI populations; thus in an embodiment,in the present invention involving Nav 1.8 the human is of ASN ancestry,eg, of CHB, CHS or JPT ancestry; or of EUR ancestry, eg, of IBS or TSIancestry. *****rs57326399 is present in humans at an average cumulativefrequency of 23% according to the 1000 Genomes Phase I database, but ishigher in ASN, EUR, CHB, CHS, JPT, CEU, GBR, IBS and TSI populations;thus in an embodiment, in the present invention involving Nav 1.8 thehuman is of ASN ancestry, eg, of CHB, CHS or JPT ancestry; or of EURancestry, eg, of CEU, GBR, IBS or TSI ancestry. +rs7630989 is present inhumans at an average cumulative frequency of 14% according to the 1000Genomes Phase I database, but is higher in AFR, ASN, ASW, LWK, YRI, CHB,CHS and JPT populations; thus in an embodiment, in the present inventioninvolving Nav 1.8 the human is of ASN ancestry, eg, of CHB, CHS or JPTancestry; or of EUR ancestry, eg, of CEU, GBR, IBS or TSI ancestry.++rs74717885 is present in humans at an average cumulative frequency of5% according to the 1000 Genomes Phase I database, but is higher in ASN,CHB, CHS and JPT populations; thus in an embodiment, in the presentinvention involving Nav 1.8 the human is of ASN ancestry, eg, of CHB,CHS or JPT ancestry. +++rs144270136 is present in humans at an averagecumulative frequency of 5% according to the 1000 Genomes Phase Idatabase, but is higher in ASN, CHB, CHS and JPT populations; thus in anembodiment, in the present invention involving Nav 1.8 the human is ofASN ancestry, eg, of CHB, CHS or JPT ancestry.In an embodiment, variations are with reference to transcriptENST00000449082 (the sequence of this transcript being incorporatedherein in its entirety).

Optionally for any mutation, the condition is a condition listed inTable 25.

In an embodiment, the human comprises a SNP selected from the SNPs incolumn 1 of Table 25; optionally also the ligand specifically binds ahuman Nav1.8 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the human comprises a Nav1.8 amino acid variationselected from the variations in column 4 of Table 25; optionally alsothe ligand specifically binds a human Nav1.8 comprising said variation.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.8-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.8protein that comprises an amino acid selected from the group consistingof L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V, S509P, 1206Mand R90W or an amino acid encoded by a SNP selected from the groupconsisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399,rs7630989, rs74717885 and rs144270136; optionally selected from thegroup consisting of L554P, A1304T, I1706V and G1662S (eg, when thecondition is neuropathic pain). These amino acid variations are found innaturally-occurring Nav1.8 variants in humans. Said human comprises anucleotide sequence encoding a Nav1.8 protein comprising said selectedamino acid.

In an example, the selected amino acid is L554P. In an example, theselected amino acid is A1304T. In an example, the selected amino acid is11706V. In an example, the selected amino acid is G1662S.

The disease or condition is, for example, any disease or conditiondescribed above in the Example relating to Nav1.7 and the disclosure ofsuch diseases and conditions are hereby also imported into this exampleto provide for possible combination of the present invention relating toNav1.8 (eg, for use in one or more claims herein). In one embodiment,the condition is a condition listed in Table 25 or a symptom of such acondition. In an example, the condition is pain or a symptom of pain,eg, a symptom of neuropathic pain. In an example, the condition isselected from the group consisting of paroxysmal pain (eg, of handsand/or feet), hand pain, foot pain, limb pain (eg, leg pain), cramp (eg,foot cramp or leg cramp), tingling (e, foot, hand or leg tingling) andpain associated with clothing contact with the human. In an example, thecondition is an abnormal threshold for warmth or cold sensation (eg, ina foot or feet). In an example, the condition is joint pain. In anexample, the condition is dry eye sensation, diahrrhoea, urinarycondition, heart palpitations or dizziness. In an example, the conditionis insensitivity to pain. In an example, the condition is episodic pain(eg, familial episodic pain).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathicpain, eg, peripheral neuropathic pain. For example, the pain is painfuldiabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminalneuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosispain, phantom limb pain, post-stroke pain, chronic back pain,osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronicinflammatory pain.

In an example, the ligand of the invention comprises an anti-humanNav1.8 binding site, wherein the binding site is a human or humanizedbinding site, eg, the binding site comprises or consists of a human orhumanized antibody variable domain or plurality of variable domains (eg,human VH/VL binding site(s)). Additionally or alternatively, the ligandcomprises one or more human antibody constant regions (eg, a humanantibody CH1, CH2, CH3 (or all of these) or Fc). In an example, theligand is an antibody that comprises human or humanized variable regionsand human constant regions (eg, bearing one or more mutations to enhanceor dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) fortreating or reducing the risk of a Nav1.8-mediated disease or conditionin a human in need thereof, the method comprising administering to saidhuman said ligand, wherein the ligand specifically binds a human Nav1.8protein that comprises an amino acid selected from the group consistingof L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V, S509P, 1206Mand R90W or an amino acid encoded by a SNP selected from the groupconsisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399,rs7630989, rs74717885 and rs144270136; optionally selected from thegroup consisting of L554P, A1304T, I1706V and G16625 (eg, when thecondition is neuropathic pain). Said human comprises a nucleotidesequence encoding a Nav1.8 protein comprising said selected amino acid.

In an example, the invention provides a method of targeting Nav1.8 in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.8protein that comprises an amino acid selected from the group consistingof L554P, A1304T, 11706V, A1073V, G16625, L1092P, I962V, 5509P, 1206Mand R90W or an amino acid encoded by a SNP selected from the groupconsisting of rs6801957, rs6795970, rs10428132, rs12632942, rs57326399,rs7630989, rs74717885 and rs144270136; optionally selected from thegroup consisting of L554P, A1304T, 11706V and G1662S (eg, when thecondition is neuropathic pain). Said human comprises a nucleotidesequence encoding a Nav1.8 protein comprising said selected amino acid.In an example, the human is suffering from or at risk of aNav1.8-mediated disease or condition. In an example, the method treatsor reduces the risk of a Nav1.8-mediated disease or condition in thehuman.

An example also provides a ligand (eg, an antibody or antibody fragment)for targeting Nav1.8 in a human, the method comprising administering tosaid human said ligand, wherein the ligand specifically binds a humanNav1.8 protein that comprises an amino acid selected from the groupconsisting of L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V,5509P, 1206M and R90W or an amino acid encoded by a SNP selected fromthe group consisting of rs6801957, rs6795970, rs10428132, rs12632942,rs57326399, rs7630989, rs74717885 and rs144270136; optionally selectedfrom the group consisting of L554P, A1304T, 11706V and G1662S (eg, whenthe condition is neuropathic pain). Said human comprises a nucleotidesequence encoding a (eg, said) Nav1.8 protein comprising said selectedamino acid. In an example, the human is suffering from or at risk of aNav1.8-mediated disease or condition. In an example, the method treatsor reduces the risk of a Nav1.8-mediated disease or condition in thehuman.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40; andwherein (ii) said human comprises a nucleotide sequence encoding saidNav1.8 protein comprising said amino acid selected from the groupconsisting of L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V,5509P, 1206M and R90W or an amino acid encoded by a SNP selected fromthe group consisting of rs6801957, rs6795970, rs10428132, rs12632942,rs57326399, rs7630989, rs74717885 and rs144270136; optionally selectedfrom the group consisting of L554P, A1304T, 11706V and G1662S (eg, whenthe condition is neuropathic pain).

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises a nucleotide sequence encoding said Nav1.8 protein comprisingsaid amino acid selected from the group consisting of L554P, A1304T,11706V, A1073V, G1662S, L1092P, I962V, 5509P, 1206M and R90W or an aminoacid encoded by a SNP selected from the group consisting of rs6801957,rs6795970, rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 andrs144270136; optionally selected from the group consisting of L554P,A1304T, 11706V and G1662S (eg, when the condition is neuropathic pain).

In an example, the ligand, antibody or fragment is for treating orreducing the risk of (or treats or reduces the risk of) pain or itching,optionally wherein the antibody is a humanized mouse or Camelid (eg,llama or camel) antibody.

In a specific embodiment, the anti-Nav1.8 ligand, antibody or fragmentalso specifically binds to another Nav selected from Nav1.1-1.9, eg, itspecifically binds to Nav1.7 or 1.9.

In a specific embodiment, the anti-Nav1.8 ligand, antibody or fragmentof present invention comprises an Fc region as per the disclosure in theNav1.7 example above.

The ligand, antibody or fragment according to the invention is fortreating or preventing or reducing the risk of (or treats or prevents orreduces the risk of), for example, any disease or condition disclosed inWO2011/051351 or U.S. Pat. No. 7,582,745, the disclosures of whichdiseases and conditions are incorporated herein by reference forpotential inclusion in one or more claims herein. Guidance on obtainingand testing antibodies can also be found in that PCT application.

Further encompassed by the invention is the use of the ligand, antibodyor fragment in the manufacture of a medicament for use to attenuate orinhibit a Nav1.8-mediated disease or disorder in a human.Nav1.8-mediated or related disorders which are treated by the ligand,antibody or fragment of the invention include, for example, a pain oritching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving Nav1.8expression and/or activity. One embodiment, among others, is a method oftreatment comprising administering an effective amount of a ligand,antibody or fragment of the invention to a patient in need thereof,wherein functional consequences of Nav1.8 activation are decreased.Another embodiment, among others, is a method of treatment comprising(i) identifying a patient demonstrating Nav1.8 expression or activity,and (ii) administering an effective amount of a ligand, antibody orfragment of the invention to the patient, wherein a functionalconsequence of Nav1.8 activation are attenuated. An effective amountaccording to the invention is an amount that modulates (e.g. decreases)the functional consequences of Nav1.8 activation so as to modulate (e.g.decrease or lessen) the severity of at least one symptom of theparticular disease or disorder being treated, but not necessarily curethe disease or disorder. Accordingly, one embodiment of the invention isa method of treating or reducing the severity of at least one symptom ofany of the disorders referred to herein, comprising administering to apatient in need thereof an effective amount of one or more ligands,antibodies or fragments of the present invention alone or in a combinedtherapeutic regimen with another appropriate medicament known in the artor described herein such that the severity of at least one symptom ofany of the disorders is reduced. Another embodiment of the invention,among others, is a method of antagonizing at least one effect of Nav1.8comprising contacting with or administering an effective amount of oneor more ligands, antibodies or fragments of the present invention suchthat said at least one effect of Nav1.8 is antagonized, e.g. the abilityof Nav1.8 to form an ion channel, such as a sodium channel

Tailoring Antibodies to Rarer Nav1.8 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1 orin the context of the Nav1.7 Example herein), the invention includes thepossibility to tailor treatment of humans further by selectingantibody-based ligands with variable domains and/or constant domainsbased on gene segments found in many humans of the ethnic populationswhere the variant TOI forms are found to meet the selection criteria ofthe invention. This also applies mutatis mutandis where the TOI is humanNav1.8, as in the present example. Thus, all disclosure herein relatingto tailoring variable and/or constant domains apply to the presentexample, relating to Nav1.8 and is combinable for use in one or moreclaims herein. All disclosure relating to variable and constant regionsand polymorphism under the subtitle “Tailoring Antibodies to RarerNav1.7 Variant Profile” in the Nav1.7 Example is also incorporated intothe present Nav1.8 Example for use mutatis mutandis with the presentNav1.8 invention and for possible inclusion in Nav1.8-limited claimsherein.

Determination of Specific Binding of Ligands of the Invention to NAV1.8Variants

The specific binding of ligands of the invention to Nav1.8 variants canbe performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human Nav1.8 protein    that comprises an amino acid selected from the group consisting of    L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V, S509P, 1206M    and R90W or an amino acid encoded by a SNP selected from the group    consisting of rs6801957, rs6795970, rs10428132, rs12632942,    rs57326399, rs7630989, rs74717885 and rs144270136; optionally    selected from the group consisting of L554P, A1304T, I1706V and    G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, 11706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.8 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human Nav1.8        protein that comprises an amino acid selected from the group        consisting of L554P, A1304T, 11706V, A1073V, G1662S, L1092P,        I962V, 5509P, 1206M and R90W or an amino acid encoded by a SNP        selected from the group consisting of rs6801957, rs6795970,        rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and        rs144270136; optionally selected from the group consisting of        L554P, A1304T, 11706V and G1662S (eg, when the condition is        neuropathic pain); wherein (i) the ligand comprises a VH domain        derived from the recombination of a human VH segment, a human D        gene segment and a human JH segment, the human VH segment        encoding the framework 1 of SEQ ID NO: 40 and wherein said human        comprises a VH gene segment encoding the framework 1 of SEQ ID        NO: 40, or the human expresses VH domains that comprise the        framework 1 of SEQ ID NO: 40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an example, the human is suffering from or at risk of a        Nav1.8-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.8-mediated disease or condition in the human.-   2. The method of clause 1, wherein said amino acid is selected from    the group consisting of L554P, A1304T, I1706V and G1662S; optionally    wherein said disease or condition is a pain disease or condition,    eg, inflammatory pain or neuropathic pain, (eg, SFN).-   3. The method of clause 1, wherein said amino acid is selected from    the group consisting of 2884A>G and 3218C>T; optionally wherein said    disease or condition is pain or is selected from dyspepsia, EPS, DPS    and gastric pain.-   4. The method of clause 1, wherein said amino acid is encoded by a    SNP selected from the group consisting of rs6801957, rs6795970 and    rs10428132; optionally wherein said disease or condition is selected    from the group consisting of Brugada syndrome, cardiac pain, cardiac    arrhythmia, heart block, ventricular fibrillation and atrial    fibrillation.-   5. The method of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid and/or a Nav1.8 protein    comprising said selected amino acid.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.8 protein comprising said selected amino acid    and/or (b) a Nav1.8 protein comprising said selected amino acid,    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.8 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.8 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136 or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.8-mediated disease    or condition (eg, asthma) in a human in need thereof, wherein the    ligand specifically binds a human Nav1.8 protein that comprises an    amino acid selected from the group consisting of L554P, A1304T,    11706V, A1073V, G1662S, L1092P, I962V, 5509P, 1206M and R90W or an    amino acid encoded by a SNP selected from the group consisting of    rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,    rs74717885 and rs144270136; optionally selected from the group    consisting of L554P, A1304T, 11706V and G1662S (eg, when the    condition is neuropathic pain);    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, 11706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.8 in a human, wherein the ligand        specifically binds a human Nav1.8 protein that comprises an        amino acid selected from the group consisting of L554P, A1304T,        11706V, A1073V, G1662S, L1092P, 1962V, 5509P, 1206M and R90W or        an amino acid encoded by a SNP selected from the group        consisting of rs6801957, rs6795970, rs10428132, rs12632942,        rs57326399, rs7630989, rs74717885 and rs144270136; optionally        selected from the group consisting of L554P, A1304T, I1706V and        G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain). In an example, the human is        suffering from or at risk of a Nav1.8-mediated disease or        condition. In an example, the method treats or reduces the risk        of a Nav1.8-mediated disease or condition in the human.-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of L554P, A1304T, 11706V and    G1662S; optionally wherein said disease or condition is a pain    disease or condition, eg, inflammatory pain or neuropathic pain,    (eg, SFN).-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 2884A>G and 3218C>T;    optionally wherein said disease or condition is pain or is selected    from dyspepsia, EPS, DPS and gastric pain.-   4. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of rs6801957, rs6795970 and    rs10428132; optionally wherein said disease or condition is selected    from the group consisting of Brugada syndrome, cardiac pain, cardiac    arrhythmia, heart block, ventricular fibrillation and atrial    fibrillation.-   5. The ligand of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid selected and/or a Nav1.8    protein comprising said selected amino acid.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.8 protein comprising said selected    amino acid and/or (b) a Nav1.8 protein comprising said selected    amino acid, optionally, wherein the determining step is performed    before administration of the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.-   10. The ligand of clause 9, wherein the assaying comprises    contacting the biological sample with at least one oligonucleotide    probe comprising a sequence of at least 10 contiguous nucleotides of    a nucleotide sequence encoding a Nav1.8 protein comprising said    selected amino acid or comprising an antisense sequence of said    contiguous nucleotides, wherein said sequence of contiguous    nucleotides comprises a nucleotide sequence encoding said selected    amino acid, thereby forming a complex when at least one nucleotide    sequence encoding the Nav1.8 protein comprising said selected amino    acid is present; and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136, or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human Nav1.8 protein    that comprises an amino acid selected from the group consisting of    L554P, A1304T, I1706V, A1073V, G1662S, L1092P, I962V, S509P, I206M    and R90W or an amino acid encoded by a SNP selected from the group    consisting of rs6801957, rs6795970, rs10428132, rs12632942,    rs57326399, rs7630989, rs74717885 and rs144270136; optionally    selected from the group consisting of L554P, A1304T, I1706V and    G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.8 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human Nav1.8        protein that comprises an amino acid selected from the group        consisting of L554P, A1304T, 11706V, A1073V, G1662S, L1092P,        I962V, 5509P, 1206M and R90W or an amino acid encoded by a SNP        selected from the group consisting of rs6801957, rs6795970,        rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and        rs144270136; optionally selected from the group consisting of        L554P, A1304T, 11706V and G1662S (eg, when the condition is        neuropathic pain); wherein (i) the ligand comprises a human        gamma-4 heavy chain constant region that comprises a Leu at        position 189 shown in SEQ ID NO: 73 or an Arg at position 289        shown in SEQ ID NO: 73 and wherein said human comprises (i) an        IGHG4*01 human heavy chain constant region gene segment, or the        human expresses antibodies comprising human gamma-4 heavy chain        constant regions comprising a Leu at position 189 shown in SEQ        ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, 11706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an example, the human is suffering from or at risk of a        Nav1.8-mediated disease or condition. In an example, the method        treats or reduces the risk of a Nav1.8-mediated disease or        condition in the human.-   2. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of L554P, A1304T, 11706V and    G1662S; optionally wherein said disease or condition is a pain    disease or condition, eg, inflammatory pain or neuropathic pain,    (eg, SFN).-   3. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 2884A>G and 3218C>T;    optionally wherein said disease or condition is pain or is selected    from dyspepsia, EPS, DPS and gastric pain.-   4. The method of any preceding clause, wherein said amino acid is    encoded by a SNP selected from the group consisting of rs6801957,    rs6795970 and rs10428132; optionally wherein said disease or    condition is selected from the group consisting of Brugada syndrome,    cardiac pain, cardiac arrhythmia, heart block, ventricular    fibrillation and atrial fibrillation.-   5. The method of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid and/or a Nav1.8 protein    comprising said selected amino acid.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.8 protein comprising said selected amino acid    and/or (b) a Nav1.8 protein comprising said selected amino acid,    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.8 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.8 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136 or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.-   22. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof-   23. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.8-mediated disease    or condition (eg, pain) in a human in need thereof, wherein the    ligand specifically binds a human Nav1.8 protein that comprises an    amino acid selected from the group consisting of L554P, A1304T,    11706V, A1073V, G1662S, L1092P, I962V, 5509P, 1206M and R90W or an    amino acid encoded by a SNP selected from the group consisting of    rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,    rs74717885 and rs144270136; optionally selected from the group    consisting of L554P, A1304T, 11706V and G1662S (eg, when the    condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).

In an alternative, paragraph 1 provides:—

-   -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.8 in a human, wherein the ligand        specifically binds a human Nav1.8 protein that comprises an        amino acid selected from the group consisting of L554P, A1304T,        11706V, A1073V, G1662S, L1092P, 1962V, 5509P, 1206M and R90W or        an amino acid encoded by a SNP selected from the group        consisting of rs6801957, rs6795970, rs10428132, rs12632942,        rs57326399, rs7630989, rs74717885 and rs144270136; optionally        selected from the group consisting of L554P, A1304T, I1706V and        G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an example, the human is suffering from or at risk of a        Nav1.8-mediated disease or condition. In an example, the method        treats or reduces the risk of a Nav1.8-mediated disease or        condition in the human.

-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of L554P, A1304T, 11706V and    G1662S; optionally wherein said disease or condition is a pain    disease or condition, eg, inflammatory pain or neuropathic pain,    (eg, SFN).

-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 2884A>G and 3218C>T;    optionally wherein said disease or condition is pain or is selected    from dyspepsia, EPS, DPS and gastric pain.

-   4. The ligand of any preceding clause, wherein said amino acid is    encoded by a SNP selected from the group consisting of rs6801957,    rs6795970 and rs10428132; optionally wherein said disease or    condition is selected from the group consisting of Brugada syndrome,    cardiac pain, cardiac arrhythmia, heart block, ventricular    fibrillation and atrial fibrillation.

-   5. The ligand of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.

-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.

-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid selected and/or a Nav1.8    protein comprising said selected amino acid.

-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.8 protein comprising said selected    amino acid and/or (b) a Nav1.8 protein comprising said selected    amino acid, optionally, wherein the determining step is performed    before administration of the antibody to the human.

-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.

-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.8 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.8 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.

-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.

-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.

-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136, or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.

-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.

-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.

-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.

-   17. The ligand of any preceding clause, wherein said disease or    condition is neuropathic pain.

-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.

-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.

-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.

-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.

-   22. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof.

-   23. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.8-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human Nav1.8 protein    that comprises an amino acid selected from the group consisting of    L554P, A1304T, 11706V, A1073V, G1662S, L1092P, I962V, S509P, I206M    and R90W or an amino acid encoded by a SNP selected from the group    consisting of rs6801957, rs6795970, rs10428132, rs12632942,    rs57326399, rs7630989, rs74717885 and rs144270136; optionally    selected from the group consisting of L554P, A1304T, I1706V and    G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.8 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human Nav1.8        protein that comprises an amino acid selected from the group        consisting of L554P, A1304T, 11706V, A1073V, G1662S, L1092P,        1962V, 5509P, 1206M and R90W or an amino acid encoded by a SNP        selected from the group consisting of rs6801957, rs6795970,        rs10428132, rs12632942, rs57326399, rs7630989, rs74717885 and        rs144270136; optionally selected from the group consisting of        L554P, A1304T, 11706V and G1662S (eg, when the condition is        neuropathic pain); wherein (i) the ligand comprises a human        gamma-2 heavy chain constant region that comprises an amino acid        selected from the group consisting of a Pro at position 72 shown        in SEQ ID NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a        Phe at position 76 shown in SEQ ID NO: 6, a Val at position 161        shown in SEQ ID NO: 6 and an Ala at position 257 shown in SEQ ID        NO: 6 and wherein said human comprises (i) an IGHG2*01 human        heavy chain constant region gene segment, or the human expresses        antibodies comprising human gamma-2 heavy chain constant regions        comprising said selected Pro at position 72 shown in SEQ ID NO:        6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76        shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6        or Ala at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an example, the human is suffering from or at risk of a        Nav1.8-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.8-mediated disease or condition in the human.-   2. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of L554P, A1304T, I1706V and    G1662S; optionally wherein said disease or condition is a pain    disease or condition, eg, inflammatory pain or neuropathic pain,    (eg, SFN).-   3. The method of any preceding clause, wherein said amino acid is    selected from the group consisting of 2884A>G and 3218C>T;    optionally wherein said disease or condition is pain or is selected    from dyspepsia, EPS, DPS and gastric pain.-   4. The method of any preceding clause, wherein said amino acid is    encoded by a SNP selected from the group consisting of rs6801957,    rs6795970 and rs10428132; optionally wherein said disease or    condition is selected from the group consisting of Brugada syndrome,    cardiac pain, cardiac arrhythmia, heart block, ventricular    fibrillation and atrial fibrillation.-   5. The method of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid and/or a Nav1.8 protein    comprising said selected amino acid.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.8 protein comprising said selected amino acid    and/or (b) a Nav1.8 protein comprising said selected amino acid,    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.8 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.8 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136 or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.-   22. The method of any preceding clause, wherein the human gamma-2    heavy chain constant region of the ligand comprises IGHG2*01 amino    acid sequence or an ADCC inactivated version thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.8-mediated disease    or condition (eg, pain) in a human in need thereof, wherein the    ligand specifically binds a human Nav1.8 protein that comprises an    amino acid selected from the group consisting of L554P, A1304T,    I1706V, A1073V, G1662S, L1092P, I962V, 5509P, 1206M and R90W or an    amino acid encoded by a SNP selected from the group consisting of    rs6801957, rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,    rs74717885 and rs144270136; optionally selected from the group    consisting of L554P, A1304T, 11706V and G1662S (eg, when the    condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, I962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, 11706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.8 in a human, wherein the ligand        specifically binds a human Nav1.8 protein that comprises an        amino acid selected from the group consisting of L554P, A1304T,        11706V, A1073V, G1662S, L1092P, 1962V, 5509P, 1206M and R90W or        an amino acid encoded by a SNP selected from the group        consisting of rs6801957, rs6795970, rs10428132, rs12632942,        rs57326399, rs7630989, rs74717885 and rs144270136; optionally        selected from the group consisting of L554P, A1304T, I1706V and        G1662S (eg, when the condition is neuropathic pain);    -   wherein (i) the ligand comprises a human gamma-2 heavy chain        constant region that comprises an amino acid selected from the        group consisting of a Pro at position 72 shown in SEQ ID NO: 6,        an Asn at position 75 shown in SEQ ID NO: 6, a Phe at position        76 shown in SEQ ID NO: 6, a Val at position 161 shown in SEQ ID        NO: 6 and an Ala at position 257 shown in SEQ ID NO: 6 and        wherein said human comprises (i) an IGHG2*01 human heavy chain        constant region gene segment, or the human expresses antibodies        comprising human gamma-2 heavy chain constant regions comprising        said selected Pro at position 72 shown in SEQ ID NO: 6, Asn at        position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in        SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or Ala        at position 257 shown in SEQ ID NO: 6; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.8 protein comprising said amino acid selected from the        group consisting of L554P, A1304T, 11706V, A1073V, G1662S,        L1092P, 1962V, 5509P, 1206M and R90W or an amino acid encoded by        a SNP selected from the group consisting of rs6801957,        rs6795970, rs10428132, rs12632942, rs57326399, rs7630989,        rs74717885 and rs144270136; optionally selected from the group        consisting of L554P, A1304T, I1706V and G1662S (eg, when the        condition is neuropathic pain).    -   In an example, the human is suffering from or at risk of a        Nav1.8-mediated disease or condition. In an example, the method        treats or reduces the risk of a Nav1.8-mediated disease or        condition in the human.-   2. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of L554P, A1304T, 11706V and    G1662S; optionally wherein said disease or condition is a pain    disease or condition, eg, inflammatory pain or neuropathic pain,    (eg, SFN).-   3. The ligand of any preceding clause, wherein said amino acid is    selected from the group consisting of 2884A>G and 3218C>T;    optionally wherein said disease or condition is pain or is selected    from dyspepsia, EPS, DPS and gastric pain.-   4. The ligand of any preceding clause, wherein said amino acid is    encoded by a SNP selected from the group consisting of rs6801957,    rs6795970 and rs10428132; optionally wherein said disease or    condition is selected from the group consisting of Brugada syndrome,    cardiac pain, cardiac arrhythmia, heart block, ventricular    fibrillation and atrial fibrillation.-   5. The ligand of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.8    protein comprising said selected amino acid selected and/or a Nav1.8    protein comprising said selected amino acid.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.8 protein comprising said selected    amino acid and/or (b) a Nav1.8 protein comprising said selected    amino acid, optionally, wherein the determining step is performed    before administration of the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.8 protein comprising said selected amino    acid.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.8 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.8 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.8 protein comprising said selected amino        acid.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.8 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 136, or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.8 protein comprising    said selected amino acid.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs138404783, rs6801957, rs6795970, rs10428132,    rs12632942, rs57326399, rs7630989, rs74717885 and rs144270136.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.-   22. The ligand of any preceding clause, wherein the human gamma-2    heavy chain constant region of the ligand comprises IGHG2*01 amino    acid sequence or an ADCC inactivated version thereof.

In any embodiment or example of the Nav1.8-focused invention, the methodoptionally reduces a symptom of neuropathic pain or inflammatory pain;or optionally the ligand is for reducing a symptom of neuropathic painor inflammatory pain.

In any embodiment or example of the Nav1.8-focused invention saidselected amino acid may be regarded as a mutation in SEQ ID NO: 135.

In an example, the human is suffering from or at risk of aNav1.8-mediated disease or condition. In an example, the method treatsor reduces the risk of a Nav1.8-mediated disease or condition in thehuman.

Example 10 Nav1.9 (SCN11A; NaN; SCN12A; SNS-2)

Nav1.9 (Sodium channel, voltage-gated, type XI, alpha subunit) is avoltage-gated sodium ion channel protein which in humans is encoded bythe SCN11A gene (OMIM: 604385). Nav1.9 has high expression levels inperipheral sensory neurons and is thought to carry the NaN current, apersistent and tetrodrotoxin (TTX)-resistant voltage-gated Na Current,involved in pain-related signalling. Nav1.9 is found within smallsensory neurons (<30 μm diameter) of dorsal root ganglia (DRG) andtrigeminal ganglia. Nav1.9 is relatively resistant to TTX, but haskinetic properties distinct from the similarly TTX-resistant Nav1.8,producing a persistent current with an activation potential of −70 mV,close to the resting membrane potential, where it may set the thresholdfor activation. A gain-of-function mutation of SCN11A was reported to belinked to congenital insensitivity to pain (Leipold et al 2013),familial episodic pain (Zhang et al 2013) and painful peripheralneuropathy (Huang et al 2014).

Human Nav1.9 polymorphism has been observed. Reference is made to thefollowing, the disclosures of which are incorporated herein by referencein their entirety:—

-   1. Waxman S G et al, Lancet Neurol. 2014 November; 13(11):1152-60.    doi: 10.1016/S1474-4422(14)70150-4; “Sodium channel genes in    pain-related disorders: phenotype-genotype associations and    recommendations for clinical use”.-   2. Hoeijmakers J G et al, “Neurosci Lett. 2014 Dec. 31. pii:    S0304-3940(14)01009-X. doi: 10.1016/j.neulet.2014.12.056; “Painful    peripheral neuropathy and sodium channel mutations”.-   3. Huang J et al, Brain. 2014 June; 137(Pt 6):1627-42. doi:    10.1093/brain/awu079. Epub 2014 Apr. 27; “Gain-of-function mutations    in sodium channel Na(v)1.9 in painful neuropathy”.-   4. Leipold E et al, Nat Genet. 2013 November; 45(11):1399-404. doi:    10.1038/ng.2767. Epub 2013 Sep. 15; “A de novo gain-of-function    mutation in SCN11A causes loss of pain perception”.-   5. Zhang X Y et al, Am J Hum Genet. 2013 Nov. 7; 93(5):957-66. doi:    10.1016/j.ajhg 0.2013.09.016. Epub 2013 Oct. 24; “Gain-of-function    mutations in SCN11A cause familial episodic pain”.-   6. Bennett D L, Brain. 2014 June; 137(Pt 6):1574-6. doi:    10.1093/brain/awu105. Epub 2014 Apr. 27; “Voltage-gated sodium    channel mutations and painful neuropathy: Na(v)1.9 joins the    family”.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.9-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.9protein. The invention also provides a corresponding ligand.

The present invention provides anti-Nav1.9 ligands; and Nav1.9-bindingor targeting ligands as described herein. The ligands have a variety ofutilities. Some of the ligands, for instance, are useful in specificbinding assays, for genotyping or phenotyping humans, affinitypurification of Nav1.9, in particular human Nav1.9 or its ligands and inscreening assays to identify other antagonists of Nav1.9 activity. Someof the ligands of the invention are useful for inhibitingNav1.9-mediated activities.

Anti-Nav1.9 ligands (eg, antibodies and anti-sense RNA) have beendeveloped based on targeting and neutralising so-called “wild-type”human Nav1.9, which is a commonly-occurring form (see, eg, SEQ ID NO:137). While such ligand may be useful for human patients harbouring thisform of human Nav1.9, the inventor considered it useful to investigatethe possibility of targeting rarer—but still naturally-occurring—formsof Nav1.9 amongst human populations. In this way, the inventor arrivedat insight into the natural occurrences and distributions of rarer humanNav1.9 forms that can serve as useful targets (at the protein or nucleicacid level) for human treatment, prophylaxis and diagnosis pertinent todiseases and conditions mediated or associated with Nav1.9 activity.This particularly provides for tailored therapies, prophylaxis anddiagnosis in humans that are devoid of the common Nav1.9 gene orprotein.

The skilled person will know that SNPs or other changes that translateinto amino acid variation can cause variability in activity and/orconformation of human targets to be addressed. This has spawned greatinterest in personalized medicine where genotyping and knowledge ofprotein and nucleotide variability is used to more effectively tailormedicines and diagnosis of patients. The invention, therefore, providesfor tailored pharmaceuticals and testing that specifically addressesrarer Nav1.9 polymorphic variant forms. Such forms or “alleles” (at thenucleotide level), comprise one or more changes at the nucleotide andamino acid levels from the corresponding common form nucleotide andamino acids sequences, ie, there are one or more non-synonymous (aka“missense”) changes at the nucleotide level that translate into one ormore corresponding changes in the protein target in humans.

Furthermore, the inventor surprisingly realised that rarer naturalforms, although present in humans at much lower frequencies than thecommon form, nevertheless are represented in multiple andethnically-diverse human populations and usually with many humanexamples per represented ethnic population. Thus, the inventor realisedthat targeting rarer forms would provide for effective treatment,prophylaxis or diagnosis across many human ethnic populations, therebyextending the utility of the present invention.

With this realisation, the inventor saw that there is significantindustrial and medical application for the invention in terms of guidingthe choice of anti-Nav1.9 ligand for administration to human patientsfor therapy and/or prophylaxis of Nav1.9-mediated or associated diseasesor conditions. In this way, the patient receives drugs and ligands thatare tailored to their needs—as determined by the patient's genetic orphenotypic makeup. Hand-in-hand with this, the invention provides forthe genotyping and/or phenotyping of patients in connection with suchtreatment, thereby allowing a proper match of drug to patient. Thisincreases the chances of medical efficacy, reduces the likelihood ofinferior treatment using drugs or ligands that are not matched to thepatient (eg, poor efficacy and/or side-effects) and avoidspharmaceutical mis-prescription and waste.

In developing this thinking, in this non-limiting example a set of humanNav1.9 variants can be determined on the basis of the followingcriteria, these being criteria that the inventor realised would providefor useful medical drugs and diagnostics to tailored need in the humanpopulation. The inventor selected some variants having at least 3 of the4 following criteria:—

Naturally-occurring human Nav1.9 variation having a cumulative humanallele frequency of 35% or less;

Naturally-occurring human Nav1.9 variation having a total human genotypefrequency of about 50% or less;

Naturally-occurring human Nav1.9 variation found in many different humanethnic populations (using the standard categorisation of the 1000Genomes Project; see Table 2 below); and

Naturally-occurring human Nav1.9 variation found in many individualsdistributed across such many different ethnic populations.

The selection can include, as an additional or alternativeconsideration, selection for nucleotide variation that produced aminoacid variation in corresponding Nav1.9 forms (ie, non-synonymousvariations), as opposed to silent variations that do not alter aminoacid residues in the target protein.

Variants can also be sourced from Ensembl, 1000 Genomes database or theliterature.

The inventor identified variants listed in Table 26 below.

TABLE 26 Nav 1.9 - Human Polymorphism & SNPs CHROMOSOMAL LOCATION AMINO(FORWARD NUCLEOTIDE ACID AMINO ACID CUMULATIVE EXAMPLE SNP/VARIANTSTRAND) CHANGE CHANGE POSITION FREQUENCY¹ CONDITIONS 1142T>C I381T 381SFN Numbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands,feet or legs) Joint pain Decreased vibration sensitivity Abnormal warmthor cold sensation K419N 419 SFN Numbness (eg, in feet) Tingling (eg, inlegs) Cramps (eg, in hands, feet or legs) Joint pain Decreased vibrationsensitivity Abnormal warmth or cold sensation A582T 582 SFN Numbness(eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs)Joint pain Decreased vibration sensitivity Abnormal warmth or coldsensation A681D 681 SFN Numbness (eg, in feet) Tingling (eg, in legs)Cramps (eg, in hands, feet or legs) Joint pain Decreased vibrationsensitivity Abnormal warmth or cold sensation A842P 842 SFN Numbness(eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feet or legs)Joint pain Decreased vibration sensitivity Abnormal warmth or coldsensation 3473T>C L1158P 1158 SFN Numbness (eg, in feet) Tingling (eg,in legs) Cramps (eg, in hands, feet or legs) Joint pain Decreasedvibration sensitivity Abnormal warmth or cold sensation F1689L 1689 SFNNumbness (eg, in feet) Tingling (eg, in legs) Cramps (eg, in hands, feetor legs) Joint pain Decreased vibration sensitivity Abnormal warmth orcold sensation 2432T>C L811P 811 Pain insensitivity Abnormal warmth orcold sensation 673C>T R225C 225 Pain Episodic pain (eg, familialepisodic pain) 2423C>G A808G 808 Pain Episodic pain (eg, familialepisodic pain) rs33985936* 3:38894643 C>T V9091 909 0.165 A paincondition rs78812474** 3:38950107 G>T R86G 86 0.061 A pain conditionrs72869687*** 3:38847244 G>A T1609I 1609 0.054 A pain conditionrs13059805**** 3:38907980 C>T G481E 481 0.013 A pain condition SFN =small fibre neuropathy or pain associated with small fibre neuropathy,SFN with large fibre involvement ¹According to Ensembl, dbSNP*rs33985936 is present in humans at an average cumulative frequency of17% according to the 1000 Genomes Phase I database, but is higher inAMR, EUR, MXL, PUR, CEU, FIN, GBR, IBS and TSI populations; thus in anembodiment, in the present invention involving Nav 1.9 the human is ofAMR ancestry, eg, of MXL or PUR ancestry; or of EUR ancestry, eg, ofCEU, FIN, GBR, IBS or TSI ancestry. **rs78812474 is present in humans atan average cumulative frequency of 6% according to the 1000 GenomesPhase I database, but is higher in AFR, LWK, YRI, MXL and GBRpopulations; thus in an embodiment, in the present invention involvingNav 1.9 the human is of AFR ancestry, eg, of LWK or YRI ancestry; or ofMXL or GBR ancestry. ***rs72869687 is present in humans at an averagecumulative frequency of 5% according to the 1000 Genomes Phase Idatabase, but is higher in AFR, ASN, ASW, LWK, YRI, CHB, CHS and JPTpopulations; thus in an embodiment, in the present invention involvingNav 1.9 the human is of AFR ancestry, eg, of ASW, LWK or YRI ancestry;or of ASN ancestry, eg, of CHB, CHS or JPT ancestry. ****rs 13059805 ispresent in humans at an average cumulative frequency of 1% according tothe 1000 Genomes Phase I database, but is higher in AMR, EUR, CLM, MXL,CEU, FIN and GBR populations; thus in an embodiment, in the presentinvention involving Nav 1.9 the human is of AMR, EUR, CLM, MXL, CEU, FINor GBR ancestry.In an embodiment, variations are with reference to transcriptENST00000302328 (the sequence of this transcript being incorporatedherein in its entirety).

Optionally for any mutation, the condition is a condition listed inTable 26.

In an embodiment, the human comprises a SNP selected from the SNPs incolumn 1 of Table 26; optionally also the ligand specifically binds ahuman Nav1.9 encoded by a nucleotide sequence comprising said SNP.

In an embodiment, the human comprises a Nav1.9 amino acid variationselected from the variations in column 4 of Table 26; optionally alsothe ligand specifically binds a human Nav1.9 comprising said variation.

In an example, the invention provides a method of treating or reducingthe risk of a Nav1.9-mediated disease or condition in a human in needthereof, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.9protein that comprises an amino acid selected from the group consistingof I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,A808G, V9091, R86G, T16091 and G481E. These amino acid variations arefound in naturally-occurring Nav1.9 variants in humans.

Said human comprises a nucleotide sequence encoding a Nav1.9 proteincomprising said selected amino acid.

Optionally in any embodiment of the Nav1.9 invention said amino acid isselected from the group consisting of I381T, K419N, A582T, A681D, A842P,L1158P, F1689L, L811P and R225C, eg, wherein the condition is a paincondition.

Optionally in any embodiment of the Nav1.9 invention said amino acid isselected from the group consisting of I381T, K419N, A582T, A681D, A842P,L1158P and F1689L, eg, wherein the condition is SFN, numbness (eg, infeet), tingling (eg, in legs), cramps (eg, in hands, feet or legs),joint pain, decreased vibration sensitivity, or abnormal warmth or coldsensation.

Optionally in any embodiment of the Nav1.9 invention said amino acid isselected from the group consisting of A808G and R225C, eg, wherein thecondition is a corresponding condition listed in Table 26.

Alternatively, in any embodiment of the Nav1.9 invention the amino acidis encoded by a SNP selected from the group consisting of rs33985936,rs78812474, rs72869687 and rs13059805, eg, wherein the condition is acorresponding condition listed in Table 26.

The disease or condition is, for example, any disease or conditiondescribed above in the Example relating to Nav1.7 and the disclosure ofsuch diseases and conditions are hereby also imported into this exampleto provide for possible combination of the present invention relating toNav1.9 (eg, for use in one or more claims herein). In one embodiment,the condition is a condition listed in Table 26 or a symptom of such acondition. In an example, the condition is pain or a symptom of pain,eg, a symptom of neuropathic pain. In an example, the condition isselected from the group consisting of paroxysmal pain (eg, of handsand/or feet), hand pain, foot pain, limb pain (eg, leg pain), cramp (eg,foot cramp or leg cramp), tingling (e, foot, hand or leg tingling) andpain associated with clothing contact with the human. In an example, thecondition is an abnormal threshold for warmth or cold sensation (eg, ina foot or feet). In an example, the condition is joint pain. In anexample, the condition is dry eye sensation, diahrrhoea, urinarycondition, heart palpitations or dizziness. In an example, the conditionis insensitivity to pain. In an example, the condition is episodic pain(eg, familial episodic pain).

In one embodiment, the pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathicpain, eg, peripheral neuropathic pain. For example, the pain is painfuldiabetic neuropathy (PDN), post-herpetic neuropathy (PHN) or trigeminalneuralgia (TN).

In an example, the pain is spinal cord injury pain, multiple sclerosispain, phantom limb pain, post-stroke pain, chronic back pain,osteoarthritis pain, cancer-associated pain or HIV-associated pain.

In one embodiment, the pain is inflammatory pain, eg, chronicinflammatory pain.

In an example, the ligand of the invention comprises an anti-humanNav1.9 binding site, wherein the binding site is a human or humanizedbinding site, eg, the binding site comprises or consists of a human orhumanized antibody variable domain or plurality of variable domains (eg,human VH/VL binding site(s)). Additionally or alternatively, the ligandcomprises one or more human antibody constant regions (eg, a humanantibody CH1, CH2, CH3 (or all of these) or Fc). In an example, theligand is an antibody that comprises human or humanized variable regionsand human constant regions (eg, bearing one or more mutations to enhanceor dampen Fc function in a human patient).

An example provides a ligand (eg, an antibody or antibody fragment) fortreating or reducing the risk of a Nav1.9-mediated disease or conditionin a human in need thereof, the method comprising administering to saidhuman said ligand, wherein the ligand specifically binds a human Nav1.9protein that comprises an amino acid selected from the group consistingof I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,A808G, V9091, R86G, T16091 and G481E. Said human comprises a nucleotidesequence encoding a Nav1.9 protein comprising said selected amino acid.

In an example, the invention provides a method of targeting Nav1.9 in ahuman, the method comprising administering to said human a ligand (eg,an antibody or antibody fragment) that specifically binds a human Nav1.9protein that comprises an amino acid selected from the group consistingof I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,A808G, V9091, R86G, T16091 and G481E. Said human comprises a nucleotidesequence encoding a Nav1.9 protein comprising said selected amino acid.In an example, the human is suffering from or at risk of aNav1.9-mediated disease or condition. In an example, the method treatsor reduces the risk of a Nav1.9-mediated disease or condition in thehuman.

An example also provides a ligand (eg, an antibody or antibody fragment)for targeting Nav1.9 in a human, the method comprising administering tosaid human said ligand, wherein the ligand specifically binds a humanNav1.9 protein that comprises an amino acid selected from the groupconsisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P,R225C, A808G, V9091, R86G, T16091 and G481E. Said human comprises anucleotide sequence encoding a (eg, said) Nav1.9 protein comprising saidselected amino acid. In an example, the human is suffering from or atrisk of a Nav1.9-mediated disease or condition. In an example, themethod treats or reduces the risk of a Nav1.9-mediated disease orcondition in the human.

In an embodiment, (i) the antibody or fragment comprises a VH domainderived from the recombination of a human VH segment, a human D genesegment and a human JH segment, the human VH segment encoding theframework 1 of SEQ ID NO: 40 and wherein said human comprises a VH genesegment encoding the framework 1 of SEQ ID NO: 40, or the humanexpresses VH domains that comprise the framework 1 of SEQ ID NO: 40; andwherein (ii) said human comprises a nucleotide sequence encoding saidNav1.9 protein comprising said amino acid selected from the groupconsisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P,R225C, A808G, V9091, R86G, T16091 and G481E.

Additionally or alternatively, in an embodiment, (i) the antibody orfragment comprises a human gamma-4 heavy chain constant region thatcomprises a Leu at position 189 shown in SEQ ID NO: 73 or an Arg atposition 289 shown in SEQ ID NO: 73 and wherein said human comprises anIGHG4*01 human heavy chain constant region gene segment, or the humanexpresses antibodies comprising human gamma-4 heavy chain constantregions comprising a Leu at position 189 shown in SEQ ID NO: 73 or anArg at position 289 shown in SEQ ID NO: 73; and wherein (ii) said humancomprises a nucleotide sequence encoding said Nav1.9 protein comprisingsaid amino acid selected from the group consisting of I381T, K419N,A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091, R86G,T16091 and G481E.

In an example, the ligand, antibody or fragment is for treating orreducing the risk of (or treats or reduces the risk of) pain or itching,optionally wherein the antibody is a humanized mouse or Camelid (eg,llama or camel) antibody.

In a specific embodiment, the anti-Nav1.9 ligand, antibody or fragmentalso specifically binds to another Nav selected from Nav1.1-1.8, eg, itspecifically binds to Nav1.7 or 1.8.

In a specific embodiment, the anti-Nav1.9 ligand, antibody or fragmentof the present invention comprises an Fc region as per the disclosure inthe Nav1.7 example above.

The ligand, antibody or fragment according to the invention is fortreating or preventing or reducing the risk of (or treats or prevents orreduces the risk of), for example, any disease or condition disclosed inWO2011/051351 or U.S. Pat. No. 7,582,745, the disclosures of whichdiseases and conditions are incorporated herein by reference forpotential inclusion in one or more claims herein. Guidance on obtainingand testing antibodies can also be found in that PCT application.

Further encompassed by the invention is the use of the ligand, antibodyor fragment in the manufacture of a medicament for use to attenuate orinhibit a Nav1.9-mediated disease or disorder in a human.Nav1.9-mediated or related disorders which are treated by the ligand,antibody or fragment of the invention include, for example, a pain oritching disease or condition.

Thus, a ligand, antibody or fragment of the invention is useful as atherapeutic agent in the treatment of a condition involving Nav1.9expression and/or activity. One embodiment, among others, is a method oftreatment comprising administering an effective amount of a ligand,antibody or fragment of the invention to a patient in need thereof,wherein functional consequences of Nav1.9 activation are decreased.Another embodiment, among others, is a method of treatment comprising(i) identifying a patient demonstrating Nav1.9 expression or activity,and (ii) administering an effective amount of a ligand, antibody orfragment of the invention to the patient, wherein a functionalconsequence of Nav1.9 activation are attenuated. An effective amountaccording to the invention is an amount that modulates (e.g. decreases)the functional consequences of Nav1.9 activation so as to modulate (e.g.decrease or lessen) the severity of at least one symptom of theparticular disease or disorder being treated, but not necessarily curethe disease or disorder. Accordingly, one embodiment of the invention isa method of treating or reducing the severity of at least one symptom ofany of the disorders referred to herein, comprising administering to apatient in need thereof an effective amount of one or more ligands,antibodies or fragments of the present invention alone or in a combinedtherapeutic regimen with another appropriate medicament known in the artor described herein such that the severity of at least one symptom ofany of the disorders is reduced. Another embodiment of the invention,among others, is a method of antagonizing at least one effect of Nav1.9comprising contacting with or administering an effective amount of oneor more ligands, antibodies or fragments of the present invention suchthat said at least one effect of Nav1.9 is antagonized, e.g. the abilityof Nav1.9 to form an ion channel, such as a sodium channel

Tailoring Antibodies to Rarer Nav1.9 Variant Profile

As outlined herein (for example, in the context of PCSK9 in Example 1 orin the context of the Nav1.7 Example herein), the invention includes thepossibility to tailor treatment of humans further by selectingantibody-based ligands with variable domains and/or constant domainsbased on gene segments found in many humans of the ethnic populationswhere the variant TOI forms are found to meet the selection criteria ofthe invention. This also applies mutatis mutandis where the TOI is humanNav1.9, as in the present example. Thus, all disclosure herein relatingto tailoring variable and/or constant domains apply to the presentexample, relating to Nav1.9 and is combinable for use in one or moreclaims herein. All disclosure relating to variable and constant regionsand polymorphism under the subtitle “Tailoring Antibodies to Rarer Nay1.7 Variant Profile” in the Nav1.7 Example is also incorporated into thepresent Nav1.9 Example for use mutatis mutandis with the present Nav1.9invention and for possible inclusion in Nav1.9-limited claims herein.

Determination of Specific Binding of Ligands of the Invention to NAV1.9Variants

The specific binding of ligands of the invention to Nav1.9 variants canbe performed using the SPR method described in Example 1.

Embodiments are provided as follows:—

Methods with VH Tailoring

-   1. A method of treating or reducing the risk of a Nav1.9-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human Nav1.9 protein    that comprises an amino acid selected from the group consisting of    I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,    A808G, V9091, R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.9 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human Nav1.9        protein that comprises an amino acid selected from the group        consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L,        L811P, R225C, A808G, V9091, R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an example, the human is suffering from or at risk of a        Nav1.9-mediated disease or condition. In an example, the method        treats or reduces the risk of a Nav1.9-mediated disease or        condition in the human.-   2. The method of clause 1, wherein said amino acid is selected from    the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P    and F1689L.-   3. The method of clause 2, wherein the condition is small fibre    neuropathy, numbness, tingling, cramps, joint pain, decreased    vibration sensitivity or abnormal warmth or cold sensation.-   4. The method of clause 1, wherein said amino acid is A808G or    R225C.-   5. The method of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.9    protein comprising said selected amino acid and/or a Nav1.9 protein    comprising said selected amino acid.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.9 protein comprising said selected amino acid    and/or (b) a Nav1.9 protein comprising said selected amino acid,    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.9 protein comprising said selected amino    acid.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.9 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.9 protein comprising said selected amino        acid.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.9 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 138 or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.9 protein comprising    said selected amino acid.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs33985936, rs78812474, rs72869687 and rs13059805.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.

Ligands with VH Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nav1.9-mediated disease    or condition (eg, asthma) in a human in need thereof, wherein the    ligand specifically binds a human Nav1.9 protein that comprises an    amino acid selected from the group consisting of I381T, K419N,    A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,    R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.9 in a human, wherein the ligand        specifically binds a human Nav1.9 protein that comprises an        amino acid selected from the group consisting of I381T, K419N,        A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,        R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a VH domain derived from the        recombination of a human VH segment, a human D gene segment and        a human JH segment, the human VH segment encoding the framework        1 of SEQ ID NO: 40 and wherein said human comprises a VH gene        segment encoding the framework 1 of SEQ ID NO: 40, or the human        expresses VH domains that comprise the framework 1 of SEQ ID NO:        40; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an example, the human is suffering from or at risk of a        Nav1.9-mediated disease or condition. In an example, the method        treats or reduces the risk of a Nav1.9-mediated disease or        condition in the human.-   2. The ligand of clause 1, wherein said amino acid is selected from    the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P    and F1689L.-   3. The ligand of clause 2, wherein the condition is small fibre    neuropathy, numbness, tingling, cramps, joint pain, decreased    vibration sensitivity or abnormal warmth or cold sensation.-   4. The ligand of clause 1, wherein said amino acid is A808G or    R225C.-   5. The ligand of any preceding clause, wherein the VH gene segment    comprised by said human is a germline VH gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.9    protein comprising said selected amino acid selected and/or a Nav1.9    protein comprising said selected amino acid.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.9 protein comprising said selected    amino acid and/or (b) a Nav1.9 protein comprising said selected    amino acid, optionally, wherein the determining step is performed    before administration of the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.9 protein comprising said selected amino    acid.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.9 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.9 protein comprising said selected amino        acid.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.9 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 138, or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.9 protein comprising    said selected amino acid.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs33985936, rs78812474, rs72869687 and rs13059805.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.

Methods with Gamma-4 Constant Region Tailoring

-   1. A method of treating or reducing the risk of a Nav1.9-mediated    disease or condition in a human in need thereof, the method    comprising administering to said human a ligand (eg, an antibody or    antibody fragment) that specifically binds a human Nav1.9 protein    that comprises an amino acid selected from the group consisting of    I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,    A808G, V9091, R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an alternative, clause 1 provides:—    -   A method of targeting Nav1.9 in a human, the method comprising        administering to said human a ligand (eg, an antibody or        antibody fragment) that specifically binds a human Nav1.9        protein that comprises an amino acid selected from the group        consisting of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L,        L811P, R225C, A808G, V9091, R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an example, the human is suffering from or at risk of a        Nav1.9-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.9-mediated disease or condition in the human.-   2. The method of clause 1, wherein said amino acid is selected from    the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P    and F1689L.-   3. The method of clause 2, wherein the condition is small fibre    neuropathy, numbness, tingling, cramps, joint pain, decreased    vibration sensitivity or abnormal warmth or cold sensation.-   4. The method of clause 1, wherein said amino acid is A808G or    R225C.-   5. The method of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The method of any preceding clause comprising, before said    administering, selecting a human comprising said nucleotide sequence    of (ii), wherein the human is the human of clause 1.-   7. The method of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.9    protein comprising said selected amino acid and/or a Nav1.9 protein    comprising said selected amino acid.-   8. The method of any preceding clause, comprising the step of    determining that the human comprises (a) the nucleotide sequence    that encodes a Nav1.9 protein comprising said selected amino acid    and/or (b) a Nav1.9 protein comprising said selected amino acid,    optionally, wherein the determining step is performed before    administration of the antibody to the human.-   9. The method of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.9 protein comprising said selected amino    acid.-   10. The method of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.9 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid is present;        and    -   detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.9 protein comprising said selected amino        acid.-   11. The method of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The method of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The method of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.9 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 138 or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.9 protein comprising    said selected amino acid.-   14. The method of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The method of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The method of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The method of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The method of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The method of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The method of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs33985936, rs78812474, rs72869687 and rs13059805.-   21. The method of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is administered by inhaled,    intravenous or subcutaneous administration and/or is comprised in an    inhalable or injectable preparation.-   22. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof.-   23. The method of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Ligands with Gamma-4 Constant Region Tailoring

-   1. A ligand (eg, an antibody or antibody fragment) for use in a    method of treating or reducing the risk of a Nay 1.9-mediated    disease or condition (eg, pain) in a human in need thereof, wherein    the ligand specifically binds a human Nav1.9 protein that comprises    an amino acid selected from the group consisting of I381T, K419N,    A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,    R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an alternative, paragraph 1 provides:—    -   A ligand (eg, an antibody or antibody fragment) for use in a        method of targeting Nav1.9 in a human, wherein the ligand        specifically binds a human Nav1.9 protein that comprises an        amino acid selected from the group consisting of I381T, K419N,        A582T, A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,        R86G, T16091 and G481E;    -   wherein (i) the ligand comprises a human gamma-4 heavy chain        constant region that comprises a Leu at position 189 shown in        SEQ ID NO: 73 or an Arg at position 289 shown in SEQ ID NO: 73        and wherein said human comprises (i) an IGHG4*01 human heavy        chain constant region gene segment, or the human expresses        antibodies comprising human gamma-4 heavy chain constant regions        comprising a Leu at position 189 shown in SEQ ID NO: 73 or an        Arg at position 289 shown in SEQ ID NO: 73; and    -   wherein (ii) said human comprises a nucleotide sequence encoding        said Nav1.9 protein comprising said amino acid selected from the        group consisting of I381T, K419N, A582T, A681D, A842P, L1158P,        F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and G481E.    -   In an example, the human is suffering from or at risk of a        Nav1.9-mediated disease or condition.    -   In an example, the method treats or reduces the risk of a        Nav1.9-mediated disease or condition in the human.-   2. The ligand of clause 1, wherein said amino acid is selected from    the group consisting of I381T, K419N, A582T, A681D, A842P, L1158P    and F1689L.-   3. The ligand of clause 2, wherein the condition is small fibre    neuropathy, numbness, tingling, cramps, joint pain, decreased    vibration sensitivity or abnormal warmth or cold sensation.-   4. The ligand of clause 1, wherein said amino acid is A808G or    R225C.-   5. The ligand of any preceding clause, wherein the constant region    gene segment comprised by said human is a germline gene segment.-   6. The ligand of any preceding clause said method comprising, before    said administering, selecting a human comprising said nucleotide    sequence of (ii), wherein the human is the human of clause 1.-   7. The ligand of any preceding clause, wherein the human has been    determined to comprise the nucleotide sequence that encodes a Nav1.9    protein comprising said selected amino acid selected and/or a Nav1.9    protein comprising said selected amino acid.-   8. The ligand of any preceding clause, said method comprising the    step of determining that the human comprises (a) the nucleotide    sequence that encodes a Nav1.9 protein comprising said selected    amino acid and/or (b) a Nav1.9 protein comprising said selected    amino acid, optionally, wherein the determining step is performed    before administration of the antibody to the human.-   9. The ligand of clause 8, wherein the step of determining comprises    assaying a biological sample from the human for a nucleotide    sequence encoding a Nav1.9 protein comprising said selected amino    acid.-   10. The ligand of clause 9, wherein the assaying comprises    -   contacting the biological sample with at least one        oligonucleotide probe comprising a sequence of at least 10        contiguous nucleotides of a nucleotide sequence encoding a        Nav1.9 protein comprising said selected amino acid or comprising        an antisense sequence of said contiguous nucleotides, wherein        said sequence of contiguous nucleotides comprises a nucleotide        sequence encoding said selected amino acid, thereby forming a        complex when at least one nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid is present;        and detecting the presence or absence of the complex, wherein        detecting the presence of the complex determines that the human        comprises the Nav1.9 protein comprising said selected amino        acid.-   11. The ligand of clause 9, wherein the assaying comprises nucleic    acid amplification and optionally one or more methods selected from    sequencing, next generation sequencing, nucleic acid hybridization,    and allele-specific amplification and/or wherein the assaying is    performed in a multiplex format.-   12. The ligand of clause 9 or 11, wherein said biological sample    comprises serum, blood, faeces, tissue, a cell, urine and/or saliva    of said human.-   13. The ligand of any preceding clause, wherein said human is    indicated as heterozygous for a nucleotide sequence encoding the    Nav1.9 protein comprising said selected amino acid, optionally,    wherein said human is further indicated as comprising the nucleotide    sequence of SEQ ID NO: 138, or said human is indicated as homozygous    for a nucleotide sequence encoding the Nav1.9 protein comprising    said selected amino acid.-   14. The ligand of any preceding clause, wherein said human is or has    been further determined to be substantially resistant to a pain or    itching treatment.-   15. The ligand of any preceding clause, wherein said human is    receiving or has received a pain or itching treatment or has reduced    responsiveness to a pain or itching treatment.-   16. The ligand of any preceding clause, wherein said disease or    condition is a pain or itching disease or condition.-   17. The ligand of any preceding clause, wherein said disease or    condition is neuropathic pain.-   18. The ligand of any preceding clause, wherein said human has been    diagnosed with at least one condition recited in clause 16 or 17.-   19. The ligand of any preceding clause, wherein said antibody or    antibody fragment treats or reduces the risk in said human of a    condition recited in clause 16 or 17.-   20. The ligand of any preceding clause, wherein the nucleotide    sequence comprises one or more SNPs selected from the group    consisting of rs33985936, rs78812474, rs72869687 and rs13059805.-   21. The ligand of any preceding clause, wherein said ligand (eg,    antibody or antibody fragment) is for inhaled, intravenous or    subcutaneous administration and/or is comprised in an inhalable or    injectable preparation.-   22. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region of the ligand comprises the amino acid    sequence of SEQ ID NO: 73 or an ADCC inactivated version thereof-   23. The ligand of any preceding clause, wherein the human gamma-4    heavy chain constant region comprises 228P and 235E.

Methods with Gamma-2 Constant Region Tailoring

-   -   1. A method of treating or reducing the risk of a        Nav1.9-mediated disease or condition in a human in need thereof,        the method comprising administering to said human a ligand (eg,        an antibody or antibody fragment) that specifically binds a        human Nav1.9 protein that comprises an amino acid selected from        the group consisting of I381T, K419N, A582T, A681D, A842P,        L1158P, F1689L, L811P, R225C, A808G, V9091, R86G, T16091 and        G481E;        -   wherein (i) the ligand comprises a human gamma-2 heavy chain            constant region that comprises an amino acid selected from            the group consisting of a Pro at position 72 shown in SEQ ID            NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at            position 76 shown in SEQ ID NO: 6, a Val at position 161            shown in SEQ ID NO: 6 and an Ala at position 257 shown in            SEQ ID NO: 6 and wherein said human comprises (i) an            IGHG2*01 human heavy chain constant region gene segment, or            the human expresses antibodies comprising human gamma-2            heavy chain constant regions comprising said selected Pro at            position 72 shown in SEQ ID NO: 6, Asn at position 75 shown            in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6,            Val at position 161 shown in SEQ ID NO: 6 or Ala at position            257 shown in SEQ ID NO: 6; and        -   wherein (ii) said human comprises a nucleotide sequence            encoding said Nav1.9 protein comprising said amino acid            selected from the group consisting of I381T, K419N, A582T,            A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,            R86G, T16091 and G481E.        -   In an alternative, clause 1 provides:—        -   A method of targeting Nav1.9 in a human, the method            comprising administering to said human a ligand (eg, an            antibody or antibody fragment) that specifically binds a            human Nav1.9 protein that comprises an amino acid selected            from the group consisting of I381T, K419N, A582T, A681D,            A842P, L1158P, F1689L, L811P, R225C, A808G, V9091, R86G,            T16091 and G481E;        -   wherein (i) the ligand comprises a human gamma-2 heavy chain            constant region that comprises an amino acid selected from            the group consisting of a Pro at position 72 shown in SEQ ID            NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at            position 76 shown in SEQ ID NO: 6, a Val at position 161            shown in SEQ ID NO: 6 and an Ala at position 257 shown in            SEQ ID NO: 6 and wherein said human comprises (i) an            IGHG2*01 human heavy chain constant region gene segment, or            the human expresses antibodies comprising human gamma-2            heavy chain constant regions comprising said selected Pro at            position 72 shown in SEQ ID NO: 6, Asn at position 75 shown            in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6,            Val at position 161 shown in SEQ ID NO: 6 or Ala at position            257 shown in SEQ ID NO: 6; and        -   wherein (ii) said human comprises a nucleotide sequence            encoding said Nav1.9 protein comprising said amino acid            selected from the group consisting of I381T, K419N, A582T,            A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,            R86G, T16091 and G481E.        -   In an example, the human is suffering from or at risk of a            Nav1.9-mediated disease or condition. In an example, the            method treats or reduces the risk of a Nav1.9-mediated            disease or condition in the human.    -   2. The method of clause 1, wherein said amino acid is selected        from the group consisting of I381T, K419N, A582T, A681D, A842P,        L1158P and F1689L.    -   3. The method of clause 2, wherein the condition is small fibre        neuropathy, numbness, tingling, cramps, joint pain, decreased        vibration sensitivity or abnormal warmth or cold sensation.    -   4. The method of clause 1, wherein said amino acid is A808G or        R225C.    -   5. The method of any preceding clause, wherein the constant        region gene segment comprised by said human is a germline gene        segment.    -   6. The method of any preceding clause comprising, before said        administering, selecting a human comprising said nucleotide        sequence of (ii), wherein the human is the human of clause 1.    -   7. The method of any preceding clause, wherein the human has        been determined to comprise the nucleotide sequence that encodes        a Nav1.9 protein comprising said selected amino acid and/or a        Nav1.9 protein comprising said selected amino acid.    -   8. The method of any preceding clause, comprising the step of        determining that the human comprises (a) the nucleotide sequence        that encodes a Nav1.9 protein comprising said selected amino        acid and/or (b) a Nav1.9 protein comprising said selected amino        acid, optionally, wherein the determining step is performed        before administration of the antibody to the human.    -   9. The method of clause 8, wherein the step of determining        comprises assaying a biological sample from the human for a        nucleotide sequence encoding a Nav1.9 protein comprising said        selected amino acid.    -   10. The method of clause 9, wherein the assaying comprises        -   contacting the biological sample with at least one            oligonucleotide probe comprising a sequence of at least 10            contiguous nucleotides of a nucleotide sequence encoding a            Nav1.9 protein comprising said selected amino acid or            comprising an antisense sequence of said contiguous            nucleotides, wherein said sequence of contiguous nucleotides            comprises a nucleotide sequence encoding said selected amino            acid, thereby forming a complex when at least one nucleotide            sequence encoding the Nav1.9 protein comprising said            selected amino acid is present; and        -   detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises the Nav1.9 protein comprising said selected            amino acid.    -   11. The method of clause 9, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   12. The method of clause 9 or 11, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.    -   13. The method of any preceding clause, wherein said human is        indicated as heterozygous for a nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid, optionally,        wherein said human is further indicated as comprising the        nucleotide sequence of SEQ ID NO: 138 or said human is indicated        as homozygous for a nucleotide sequence encoding the Nav1.9        protein comprising said selected amino acid.    -   14. The method of any preceding clause, wherein said human is or        has been further determined to be substantially resistant to a        pain or itching treatment.    -   15. The method of any preceding clause, wherein said human is        receiving or has received a pain or itching treatment or has        reduced responsiveness to a pain or itching treatment.    -   16. The method of any preceding clause, wherein said disease or        condition is a pain or itching disease or condition.    -   17. The method of any preceding clause, wherein said disease or        condition is neuropathic pain.    -   18. The method of any preceding clause, wherein said human has        been diagnosed with at least one condition recited in clause 16        or 17.    -   19. The method of any preceding clause, wherein said antibody or        antibody fragment treats or reduces the risk in said human of a        condition recited in clause 16 or 17.    -   20. The method of any preceding clause, wherein the nucleotide        sequence comprises one or more SNPs selected from the group        consisting of rs33985936, rs78812474, rs72869687 and rs13059805.    -   21. The method of any preceding clause, wherein said ligand (eg,        antibody or antibody fragment) is administered by inhaled,        intravenous or subcutaneous administration and/or is comprised        in an inhalable or injectable preparation.    -   22. The method of any preceding clause, wherein the human        gamma-2 heavy chain constant region of the ligand comprises        IGHG2*01 amino acid sequence or an ADCC inactivated version        thereof.

Ligands with Gamma-2 Constant Region Tailoring

-   -   1. A ligand (eg, an antibody or antibody fragment) for use in a        method of treating or reducing the risk of a Nav1.9-mediated        disease or condition (eg, pain) in a human in need thereof,        wherein the ligand specifically binds a human Nav1.9 protein        that comprises an amino acid selected from the group consisting        of I381T, K419N, A582T, A681D, A842P, L1158P, F1689L, L811P,        R225C, A808G, V9091, R86G, T16091 and G481E;        -   wherein (i) the ligand comprises a human gamma-2 heavy chain            constant region that comprises an amino acid selected from            the group consisting of a Pro at position 72 shown in SEQ ID            NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at            position 76 shown in SEQ ID NO: 6, a Val at position 161            shown in SEQ ID NO: 6 and an Ala at position 257 shown in            SEQ ID NO: 6 and wherein said human comprises (i) an            IGHG2*01 human heavy chain constant region gene segment, or            the human expresses antibodies comprising human gamma-2            heavy chain constant regions comprising said selected Pro at            position 72 shown in SEQ ID NO: 6, Asn at position 75 shown            in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6,            Val at position 161 shown in SEQ ID NO: 6 or Ala at position            257 shown in SEQ ID NO: 6; and        -   wherein (ii) said human comprises a nucleotide sequence            encoding said Nav1.9 protein comprising said amino acid            selected from the group consisting of I381T, K419N, A582T,            A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,            R86G, T16091 and G481E.        -   In an alternative, paragraph 1 provides:—        -   A ligand (eg, an antibody or antibody fragment) for use in a            method of targeting Nav1.9 in a human, wherein the ligand            specifically binds a human Nav1.9 protein that comprises an            amino acid selected from the group consisting of I381T,            K419N, A582T, A681D, A842P, L1158P, F1689L, L811P, R225C,            A808G, V9091, R86G, T16091 and G481E;        -   wherein (i) the ligand comprises a human gamma-2 heavy chain            constant region that comprises an amino acid selected from            the group consisting of a Pro at position 72 shown in SEQ ID            NO: 6, an Asn at position 75 shown in SEQ ID NO: 6, a Phe at            position 76 shown in SEQ ID NO: 6, a Val at position 161            shown in SEQ ID NO: 6 and an Ala at position 257 shown in            SEQ ID NO: 6 and wherein said human comprises (i) an            IGHG2*01 human heavy chain constant region gene segment, or            the human expresses antibodies comprising human gamma-2            heavy chain constant regions comprising said selected Pro at            position 72 shown in SEQ ID NO: 6, Asn at position 75 shown            in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6,            Val at position 161 shown in SEQ ID NO: 6 or Ala at position            257 shown in SEQ ID NO: 6; and        -   wherein (ii) said human comprises a nucleotide sequence            encoding said Nav1.9 protein comprising said amino acid            selected from the group consisting of I381T, K419N, A582T,            A681D, A842P, L1158P, F1689L, L811P, R225C, A808G, V9091,            R86G, T16091 and G481E.        -   In an example, the human is suffering from or at risk of a            Nav1.9-mediated disease or condition. In an example, the            method treats or reduces the risk of a Nav1.9-mediated            disease or condition in the human.    -   2. The ligand of clause 1, wherein said amino acid is selected        from the group consisting of I381T, K419N, A582T, A681D, A842P,        L1158P and F1689L.    -   3. The ligand of clause 2, wherein the condition is small fibre        neuropathy, numbness, tingling, cramps, joint pain, decreased        vibration sensitivity or abnormal warmth or cold sensation.    -   4. The ligand of clause 1, wherein said amino acid is A808G or        R225C.    -   5. The ligand of any preceding clause, wherein the constant        region gene segment comprised by said human is a germline gene        segment.    -   6. The ligand of any preceding clause said method comprising,        before said administering, selecting a human comprising said        nucleotide sequence of (ii), wherein the human is the human of        clause 1.    -   7. The ligand of any preceding clause, wherein the human has        been determined to comprise the nucleotide sequence that encodes        a Nav1.9 protein comprising said selected amino acid selected        and/or a Nav1.9 protein comprising said selected amino acid.    -   8. The ligand of any preceding clause, said method comprising        the step of determining that the human comprises (a) the        nucleotide sequence that encodes a Nav1.9 protein comprising        said selected amino acid and/or (b) a Nav1.9 protein comprising        said selected amino acid, optionally, wherein the determining        step is performed before administration of the antibody to the        human.    -   9. The ligand of clause 8, wherein the step of determining        comprises assaying a biological sample from the human for a        nucleotide sequence encoding a Nav1.9 protein comprising said        selected amino acid.    -   10. The ligand of clause 9, wherein the assaying comprises        -   contacting the biological sample with at least one            oligonucleotide probe comprising a sequence of at least 10            contiguous nucleotides of a nucleotide sequence encoding a            Nav1.9 protein comprising said selected amino acid or            comprising an antisense sequence of said contiguous            nucleotides, wherein said sequence of contiguous nucleotides            comprises a nucleotide sequence encoding said selected amino            acid, thereby forming a complex when at least one nucleotide            sequence encoding the Nav1.9 protein comprising said            selected amino acid is present; and        -   detecting the presence or absence of the complex, wherein            detecting the presence of the complex determines that the            human comprises the Nav1.9 protein comprising said selected            amino acid.    -   11. The ligand of clause 9, wherein the assaying comprises        nucleic acid amplification and optionally one or more methods        selected from sequencing, next generation sequencing, nucleic        acid hybridization, and allele-specific amplification and/or        wherein the assaying is performed in a multiplex format.    -   12. The ligand of clause 9 or 11, wherein said biological sample        comprises serum, blood, faeces, tissue, a cell, urine and/or        saliva of said human.    -   13. The ligand of any preceding clause, wherein said human is        indicated as heterozygous for a nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid, optionally,        wherein said human is further indicated as comprising the        nucleotide sequence of SEQ ID NO: 138, or said human is        indicated as homozygous for a nucleotide sequence encoding the        Nav1.9 protein comprising said selected amino acid.    -   14. The ligand of any preceding clause, wherein said human is or        has been further determined to be substantially resistant to a        pain or itching treatment.    -   15. The ligand of any preceding clause, wherein said human is        receiving or has received a pain or itching treatment or has        reduced responsiveness to a pain or itching treatment.    -   16. The ligand of any preceding clause, wherein said disease or        condition is a pain or itching disease or condition.    -   17. The ligand of any preceding clause, wherein said disease or        condition is neuropathic pain.    -   18. The ligand of any preceding clause, wherein said human has        been diagnosed with at least one condition recited in clause 16        or 17.    -   19. The ligand of any preceding clause, wherein said antibody or        antibody fragment treats or reduces the risk in said human of a        condition recited in clause 16 or 17.    -   20. The ligand of any preceding clause, wherein the nucleotide        sequence comprises one or more SNPs selected from the group        consisting of rs33985936, rs78812474, rs72869687 and rs13059805.    -   21. The ligand of any preceding clause, wherein said ligand (eg,        antibody or antibody fragment) is for inhaled, intravenous or        subcutaneous administration and/or is comprised in an inhalable        or injectable preparation.    -   22. The ligand of any preceding clause, wherein the human        gamma-2 heavy chain constant region of the ligand comprises        IGHG2*01 amino acid sequence or an ADCC inactivated version        thereof.

In any embodiment or example of the Nav1.9-focused invention, the methodoptionally reduces a symptom of neuropathic pain or inflammatory pain;or optionally the ligand is for reducing a symptom of neuropathic painor inflammatory pain.

In any embodiment or example of the Nav1.9-focused invention saidselected amino acid may be regarded as a mutation in SEQ ID NO: 137.

In an example, the human is suffering from or at risk of aNav1.9-mediated disease or condition. In an example, the method treatsor reduces the risk of a Nav1.9-mediated disease or condition in thehuman.

I claim:
 1. A method of treating or reducing the risk of anaemia in ahuman haemodialysis patient, the method comprising administering a trap,an antibody or an antibody fragment that binds specifically to BMP6 tothe human haemodialysis patient, wherein the trap, antibody or antibodyfragment comprises a human kappa chain constant region comprising a Valat position 84 shown in SEQ ID NO: 93 or a Cys at position 87 shown inSEQ ID NO: 9; and wherein (i) the human comprises a TMPRSS6 nucleotidesequence comprising a SNP rs855791; rs2543519; rs2235324; or rs1421312;and (ii) the human comprises and expresses an IGKC1*01 human kappa chainconstant region gene segment, or the human expresses antibodiescomprising human kappa chain constant regions comprising a Valcorresponding to position 84 shown in SEQ ID NO: 93 or a Cys at position87 shown in SEQ ID NO:
 93. 2. The method of claim 1, wherein the humanhaemodialysis patient is homozygous for said selected SNP.
 3. The methodof claim 1, wherein the trap, antibody or antibody fragment comprises anIGKC1*01 human kappa chain constant region.
 4. The method of claim 1,comprising, before said administering, selecting a human haemodialysispatient comprising said TMPRSS6 nucleotide sequence, wherein the humanis the human of claim
 1. 5. The method of claim 1, wherein the humanhaemodialysis patient has been determined to comprise said TMPRSS6nucleotide sequence.
 6. The method of claim 1, comprising the step ofdetermining that the human haemodialysis patient comprises said TMPRSS6nucleotide sequence, optionally, wherein the determining step isperformed before administration of the trap, antibody, or antibodyfragment to the human.
 7. The method of claim 6, wherein the step ofdetermining comprises assaying a biological sample from the humanhaemodialysis patient for a nucleotide sequence encoding said SNP. 8.The method of claim 7, wherein the assaying comprises contacting thebiological sample with at least one oligonucleotide probe comprising asequence of at least 10 contiguous nucleotides that can specificallyhybridize to and identify in the biological sample a nucleotide sequencecomprising said SNP or that specifically hybridizes to an antisense ofsaid sequence, wherein said nucleic acid hybridizes to said SNP orhybridizes to an antisense sequence thereby forming a complex when atleast one nucleotide sequence comprising said SNP is present; and/or atleast one oligonucleotide probe comprising a sequence of at least 10contiguous nucleotides of a nucleotide sequence comprising said SNP orcomprising an antisense sequence of said contiguous nucleotides, whereinsaid sequence of contiguous nucleotides comprises said SNP therebyforming a complex when the nucleotide sequence comprising said SNP ispresent; and detecting the presence or absence of the complex, whereindetecting the presence of the complex determines that the humancomprises a nucleotide sequence encoding TMPRSS6 and comprising saidSNP.
 9. The method of claim 7, wherein the assaying comprises nucleicacid amplification and optionally one or more methods selected fromsequencing, next generation sequencing, nucleic acid hybridization, andallele-specific amplification or wherein the assaying is performed in amultiplex format.
 10. The method of claim 1, wherein said humanhaemodialysis patient is or has been further determined to besubstantially resistant to an anaemia treatment.
 11. The method of claim1, wherein said human haemodialysis patient is receiving or has receivedan anaemia treatment or has reduced responsiveness to an anaemiatreatment.
 12. The method of claim 7, wherein said biological samplecomprises serum, blood, faeces, tissue, a cell, urine or saliva of saidhuman.
 13. The method of claim 1, wherein said trap, antibody orantibody fragment is administered by intravenous or subcutaneousinjection or is comprised in an injectable preparation.
 14. The methodof claim 1, wherein the trap, antibody or antibody fragment is a humantrap, antibody, or antibody fragment.
 15. The method of claim 1, whereinsaid human haemodialysis patient is or has been further determined to besubstantially resistant to an EPO anaemia treatment.
 16. The method ofclaim 1, wherein said human haemodialysis patient is receiving or hasreceived an EPO anaemia treatment or has reduced responsiveness to anEPO anaemia treatment.
 17. A method of treating or reducing the risk ofanaemia in a human haemodialysis patient in need thereof, theimprovement comprising: administering to said patient an anti-BMP6 trap,antibody or antibody fragment to specifically bind in the patient aBMP6; wherein the trap, antibody, or antibody fragment comprises a humankappa chain constant region comprising a Val at position 84 shown in SEQID NO: 93 or a Cys at position 87 shown in SEQ ID NO: 93, and whereinsaid patient comprises: (i) TMPRSS6 nucleotide sequence comprising a SNPrs855791; rs2543519; rs2235324; or rs1421312; and (ii) a constant regiongene segment encoding said selected constant region; or wherein saidpatient expresses antibodies comprising said selected constant region.18. The method of claim 17, wherein the human haemodialysis patient ishomozygous for said selected SNP.
 19. The method of claim 17, whereinthe trap, antibody, or antibody fragment comprises an IGKC1*01 humankappa chain constant region.
 20. The method of claim 17, comprising,before said administering, selecting a human haemodialysis patientcomprising said TMPRSS6 nucleotide sequence, wherein the human is thehuman of claim
 17. 21. The method of claim 17, wherein the humanhaemodialysis patient has been determined to comprise said TMPRSS6nucleotide sequence.
 22. The method of claim 17, comprising the step ofdetermining that the human haemodialysis patient comprises said TMPRSS6nucleotide sequence, optionally, wherein the determining step isperformed before administration of the trap, antibody, or antibodyfragment to the human.
 23. The method of claim 17, wherein the step ofdetermining comprises assaying a biological sample from the humanhaemodialysis patient for a nucleotide sequence encoding said SNP. 24.The method of claim 23, wherein the assaying comprises contacting thebiological sample with at least one oligonucleotide probe comprising asequence of at least 10 contiguous nucleotides that can specificallyhybridize to and identify in the biological sample a nucleotide sequencecomprising said SNP or that specifically hybridizes to an antisense ofsaid sequence, wherein said nucleic acid hybridizes to said SNP orhybridizes to an antisense sequence thereby forming a complex when atleast one nucleotide sequence comprising said SNP is present; and/or atleast one oligonucleotide probe comprising a sequence of at least 10contiguous nucleotides of a nucleotide sequence comprising said SNP orcomprising an antisense sequence of said contiguous nucleotides, whereinsaid sequence of contiguous nucleotides comprises said SNP therebyforming a complex when the nucleotide sequence comprising said SNP ispresent; and detecting the presence or absence of the complex, whereindetecting the presence of the complex determines that the humancomprises a nucleotide sequence encoding TMPRSS6 and comprising saidSNP.
 25. The method of claim 23, wherein the assaying comprises nucleicacid amplification and optionally one or more methods selected fromsequencing, next generation sequencing, nucleic acid hybridization, andallele-specific amplification or wherein the assaying is performed in amultiplex format.
 26. The method of claim 17, wherein said humanhaemodialysis patient is or has been further determined to besubstantially resistant to an anaemia treatment.
 27. The method of claim17, wherein said human haemodialysis patient is receiving or hasreceived an anaemia treatment or has reduced responsiveness to ananaemia treatment.
 28. The method of claim 23, wherein said biologicalsample comprises serum, blood, faeces, tissue, a cell, urine or salivaof said human.
 29. The method of claim 17, wherein said trap, antibody,or antibody fragment is administered by intravenous or subcutaneousinjection or is comprised in an injectable preparation.
 30. The methodof claim 17, wherein the trap, antibody, or antibody fragment is a humantrap, antibody, or antibody fragment.